bluuebunny/crossref_metadata_2025 huggingface dataset:
Proliferation of chick embryo neuroblasts grown in the presence of horse serum requires exogenous transferrin
Authors: Barakat‐Walter I. , Deloulme J. C. , Sensenbrenner M. , Labourdette G.
Published: January 2005

We have previously shown that neuroblasts from cerebral hemispheres of 6‐day‐old chick embryos are able to proliferate when grown in the presence of fetal calf serum. We report here that in the presence of horse serum alone the proliferative rate of neuroblasts is strongly reduced. A high proliferative rate is restored upon the addition of bovine transferrin and to a lesser extent with added FeSO4 or hemin. These findings suggest that the transferrin of horse serum cannot be used by chick neuroblasts in vitro, while bovine transferrin exogenously added is active in promoting cell proliferation. We propose that the stimulatory activity of the fetal calf serum is due to bovine transferrin, since when this serum is fractionated by gel filtration, the fractions that stimulate the proliferation of neuroblasts grown in the presence of horse serum are located in the molecular weight area of transferrin, and they do contain transferrin as seen by immunoblotting with a specific anti‐transferrin antibody.
Brief chronic effects of lithium administration on rat brain phosphoinositides and phospholipids
Authors: Navidi M. , Yoa F.‐G. , Sun G. Y.
Published: January 2005

Lithium is known to exert its biochemical action on cells and tissues by inhibiting the enzymic conversion of inositol monophosphates to inositol. However, it is not clear whether this inhibitory action may lead to changes in the de novo biosynthesis of phosphatidylinositol and its phosphorylated derivatives. This biosynthetic scheme may have an important bearing with regard to the receptor‐mediated signal transduction mechanism involving hydrolysis of polyphosphoinositides and release of inositol trisphosphate as second messenger for mobilization of intracellular calcium. In this study, the effects of brief chronic lithium administration on metabolism of brain phosphoinositides and other phospholipids were examined using the radiotracer technique with 32Pi as precursor. Sprague Dawley rats that were treated with lithium (3‐4 meq/kg body wt) twice daily for 2‐6 days consistently indicated an increase in the labeling of phosphatidylinositol 4,5‐bisphosphates and a decrease in labeling of phosphatidylinositols and phosphatidylethanolamines. These phosphoiipid changes were found in both cortex and hippocampus and appeared to occur primarily in the synaptosomal fraction. Although the extent of the phosphoiipid changes could vary depending on both duration and dose levels of the lithium administered, these results demonstrated subtle effects of lithium on depressing the biosynthesis of phosphatidylinositol as well as phosphatidylethanolamine but perhaps a compensative increase in the synthesis of the phosphatidylinositol 4,5‐bisphosphates.
Characteristics of excitatory amino acid uptake in cultures from neurons and glia from the retina
Authors: Somohano F. , López‐Colomé A. M.
Published: January 2005

3H‐D‐Aspartate uptake was biochemically characterized in cultures from chick retina enriched in glial (Müller) cells or neurons during progressive days in vitro (DIV). In the neuronal cultures a high‐affinity, Na+‐dependent system was found with Km = 8–13 μM and pharmacological characteristics in agreement with those of reuptake systems in other regions of the CNS. The uptake system in glial cells showed a lower affinity, with Km = 100–135 μM. In both cases, uptake wastemperature and energy dependent. A sharp increase in the Vmax of uptake was observed in both neuronal and glial cultures at 5 DIV, at which time morphologically mature synapses have been shown to be present in retinal cultures. A parallel increase in the pharmacological specificity of the uptake system in neuronal cultures was observed, with a rise in the efficiency of D‐Asp, L‐Asp, L‐Glu, and DL‐asp‐ β‐hydroxamate for inhibiting 3H‐D‐As‐partate uptake. Results suggest the possibility of re‐uptake participating in the regulation of extracellular glutamate concentration during development.
Transfer of 45Ca and 36Cl at the blood‐nerve barrier of the sciatic nerve in rats fed low or high calcium diets
Authors: Wadhwani K. C. , Murphy V. A. , Rapoport S. I.
Published: January 2005

Unidirectional fluxes of 45Ca, 36C1, of [3H]mannitol from blood into the sciatic nerve and cerebral cortex were determined from 5‐ and 15‐min uptakes of these tracers after an intravenous (i.v.) bolus injection in awake rats. Rats were fed diets for 8 wk, that had either a low (0.01%wt/wt), normal (0.67%), or high (3%) Ca content. Plasma[Ca] was 32% less and 11% more in rats fed low (LOCA) and high Ca diets (HICA), respectively, than in rats fed a normal Ca diet (CONT). The mean permeability‐sur‐face area product (PA) of 45Ca at the blood‐nerve barrier was about eightfold higher than at the blood‐brain barrier in the same animals and did not differ significantly between groups (<0.05). Mean PA ratios of 45Ca/36Cl barriers in CONT rats, 0.52±0.04 and 0.40±0.02, respectively, were not significantly different from corresponding ratios in LOCA and HICA groups, and corresponded to the aqueous limiting diffusion ratio (0.45), Our results show no evidence for concentration‐dependent transport of Ca over a plasma [Ca] range of 0.8‐1.4 mmol/liter at the blood‐nerve barrier of the rat peripheral nerve, and suggest that Ca and Cl exchange slowly between nerve and blood via paracellular pathways.
In vitro changes in the fine structure and protein composition of light myelin fractions isolated from guinea pig brain
Authors: Nunzi M. G. , Chan K.‐F. J. , Webster Henry deF.
Published: January 2005

To find out if in vitro maintenance produces changes in the electron microscopic appearance, protein composition, and phosphorylation properties of guinea pig CNS myelin fractions, we incubated them for 10 min, 4 hr, 24 hr, and 48 hr in phosphate‐buffered saline (pH 7.4) or in 20 mM Hepes, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM dithiothreitol, and 20 mM NaCl at 4 and 30°C. Aliquots were processed for electron microscopic study, were analyzed for protein content by gel electrophoresis, and were assayed for endogenous protein phosphorylation. Before incubation, electron micrographs of fractions contained two types of multilamellar whorls with the periodicity of CNS myeiin sheaths. The first type of whorl was separated from nearby whorls; the other type had surface lamellae that were connected to other multilayered membrane fragments. After incubation at 4°C for 24 hr, the number of both types of multilamellar whorls in micrographs had increased approximately 3‐ to 4‐fold. Counts per unit area showed that the observed increase was both time‐ and temperature‐dependent. In aliquots studied by gel elect, ophoresis, only minor degradation of myeiin proteins was observed. The endogenous protein phosphorylation properties of the myeiin fragments also remained functional, suggesting that the activities of protein phosphotransferases were not altered. We conclude that the incubation conditions described here favor interactions of proteins and lipids that lead to the formation of multi‐layered aggregates of CNS myeiin membranes.
Increased release of an amyloidogenic C‐terminal Alzheimer amyloid precursor protein fragment from stressed PC‐12 cells
Authors: Baskin F. , Rosenberg R. N. , Greenberg B. D.
Published: January 2005

Amyloid plaques, found in characteristically large numbers in specific brain areas of Alzheimer's disease (AD) and Down's Syndrome (DS) patients, are composed of a 417–43 amino acid peptide, A4, derived from a transmembrane glycoprotein, amyloid precursor protein (APP). In transformed cells APP has been shown to be cleaved within the extracellular portion of the A4 region causing the release of 100–120 kDa soluble N‐terminal APP products. If this cleavage occurs in human tissue, neither the soluble product nor the remaining 10–12 kDa transmembrane fragment could be further degraded to yield A4. It has been hypothesized that an alternate APP cleavage product containing the intact A4 region is released in increased amounts in AD and DS brain where subsequent extracellular degradation produces the amy‐loidogenic A4 peptide.

In support of this hypothesis, we have found that PC‐12 cells maintained in serum‐free media with or without additional injurious agents release a 60 kDa protein which has been detected by immunoprecipitation and immunoblot analyses with 9 antisera elicited by 4 distinct peptides within the carboxyl‐terminal half of APP. Six of these antisera, elicited by peptides corresponding to the carboxyl‐terminal 20 amino acids of APP, or the A4 peptide itself, do not bind the normally released 120 kDa APP product which is detected by 11 other antisera elicited by peptides with the N‐terminal portion of APP. Controls in which two 60 kDa‐detecting antisera were preabsorbed with the peptides used to elicit them, produced markedly reduced 60 kd bands on immunoblots. Thus, the 60 kDa protein appears to be the first reported APP product released from neural cells which, containing the intact A4 region, is potentially amyloidogenic.

Similar analyses of proteins released into the serum‐free media of PC‐12 cells maintained in 0.0030.005% SDS, 60 mM chloroquine, or other membrane damaging or lysosomal inhibiting agents revealed increased 60 kDa band intensities. Cultures grown with other classes of injurious agents did not demonstrate increased release. Together these data indicate that neural cells maintained under selected stressful or injurious conditions release increased quantities of a potentially amyloidogenic APP product, supporting the importance of a similar induction of alternative APP cleavage in AD and DS brain amy‐loidogenesis.
Possible neurotransmitter role of noradrenaline in the teleost retina
Authors: Jaffe E. H. , Urbina M. , Drujan B. D.
Published: January 2005

The role of dopamine as a neurotransmitter in the retina of different species has been clearly established; however, there is still some controversy as to whether noradrenaline (NA) is present as a neurotransmitter in this tissue. In this study, we show that, under controlled conditions, NA is present in the retina of goldfish at a concentration of 0.15±0.03 ng/ mg protein and its biosynthetic enzyme, dopamine‐β‐hydroxylase shows an activity of 2.5±0.2 pmol NA/hr/mg protein. The amount of NA increases to 1.88±0.24 ng/mg protein in light adapted animals and decreases to undetectable levels in dark adapted ones. By contrast, dopamine±β‐hydroxylase levels are not affected by changes in light conditions. This finding provides further evidence in favor of a neurotransmitter role for NA in vertebrate retina.
Spatial distribution of myelin basic protein mRNA and polypeptide in Quaking oligodendrocytes in culture
Authors: Barbarese E.
Published: January 2005

In the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of Oligodendrocytes were established from affected and control animals. The cytoarchitecture of the Oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control Oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP poly‐peptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture.
Expression of a novel gene product by transplants of genetically modified primary fibroblasts in the central nervous system
Authors: Doering L. C. , Chang P. L.
Published: January 2005

Primary fibroblasts initiated from skin biopsies of Wistar rats were transfected with a plasmid that encodes the human growth hormone and the neomycin resistance genes. Cell clones selected for G418 resistance and expressing high levels of human growth hormone were propagated in vitro and subsequently transplanted into the cerebral cortex of adult allogeneic rats. Grafts were examined by immunocytochemistry at weekly intervals up to 2 months. Fibroblasts in the transplants survived and expressed growth hormone clearly up to 4 weeks, but showed reduced expression at 6 to 8 weeks. Significant levels of human growth hormone were also detected by radioimmunoassay in the serum of the host rats up to 1 month. The experiments demonstrate that primary fibroblasts can be genetically modified to deliver a new gene product into the central nervous system, and the gene product can pass the blood‐brain barrier to enter the systemic circulation. This model illustrates the potential to introduce desired products into the brain through the genetic alteration of autologous primary fibroblasts.
Calcium‐activated neutral proteinase (CANP; calpain) activity in Schwann cells: Immunofluorescence localization and compartmentation of μ‐ and mCANP
Authors: Banik N. L. , DeVries G. H. , Neuberger T. , Russell T. , Chakrabarti A. K. , Hogan E. L.
Published: January 2005

Calcium‐activated neutral proteinase (CANP) activity was determined in cytosolic and membranous subcellular fractions of transformed Schwann cells (tSc). The μM and mM Ca2+ ‐sensitive (μ‐and mCANP) forms of CANP were separated by DEAE and phenyl Sepharose column chromatography, the latter step enabling removal of the endogenous inhibitor calpastatin. The tSc contained more μCANP than the mM isoform. More than 75% of mCANP activity was membrane‐associated and 20% was cytosolic. In contrast, approximately 80% of μCANP was cytosolic and 15% was membranous. Triton X‐100 stimulated activity of the whole homogenate and of the membrane pellet but did not stimulate CANP activity in the cytosolic fraction. Immunohistochemical distribution of mM enzyme was studied in both fixed and permeabilized tSc with cytosolic (anti‐cyt‐mCANP) and myelin (anti‐my‐mCANP) antibodies. Live cells (non‐permeabilized) stained with anti‐my‐mCANP had a single filamentous ring circumscribing individual cells. Permeabilized cells treated with anti‐my‐mCANP had immu‐noreactive deposits throughout the intracellular space but sparing the perinuclear region. No immunohistochemical staining was detected when live cells were exposed to anti‐cyt‐mCANP whereas permeabilized cells had extensive intracellular staining with the most intense immunoreactivity in the perinuclear region. Our results indicate that both forms of CANP are present in tSc and that the activity of most of the μCANP is cytosolic while mCANP is particulate.
Distribution of protein kinase C isozymes in rat optic nerves
Authors: Komoly S. , Liu Y. , Webster H. deF. , Chan K.‐F. J.
Published: January 2005

Light (LM) and electron (EM) microscopic immunocytochemical methods were used to study the distribution of protein kinase C (PKC) isozymes in adult rat optic nerves. In cryostat and vibratome sections examined by LM, type II (βrpar; isozyme was localized almost exclusively in the axons. In the EM, immunoreaction products were found to associate with microtubules and neurofilaments. The inner surface of axonal membranes were occasionally stained. Analysis of PKC isozyme composition of the optic nerves by using immunoblot techniques revealed that type II (β) isozyme accounted for approximately 80% of the total immunoreactivity. By contrast, type III (α) isozyme, which accounted for the remaining 20% of PKC, was found mainly in the astrocytes. Astrocytic processes next to blood vessels and between myelinated axons were stained. In the EM, immunoreaction products were found in the cytoplasm and along astroglial filaments. Segments of plasma membranes also were stained; but nuclei were unstained. Adult glial cells were not stained by an antibody to type II (β) isozyme except for the occurrence of a few punctate cytoplasmic densities in occasional astrocytes. Very faint or no immunostaining was observed in sections treated with a monoclonal antibody to type I (α) isozyme. Immunoblot analyses also did not reveal this subspecies. The absence of type I (α) isozyme in optic nerves is not due to a down‐regulation of the enzyme during development. In developing (5 and 11 day) rats, immunoreactivity of protein kinase C was very faint or absent. After 15 days, reaction products of both type III (α) and type II (β) isozymes were found throughout the nerve. These findings suggest that type II (β) isozyme may be involved in axonal transport whereas type III (α) isozyme may play a role in some astrocyte functions in mature optic nerves.
Identification of transcriptionally regulated genes after sciatic nerve injury
Authors: Leon M. De , Welcher A. A. , Suter U. , Shooter E. M.
Published: January 2005

Mammalian peripheral nerve fibres can regenerate after injury. In an attempt toward a better understanding of the underlying molecular events, we have isolated novel and known rat cDNA sequences, the expression of which are regulated during sciatic nerve regeneration. For this purpose, cDNA libraries were constructed from either the nerve segment distal to the crush site or the corresponding contralateral uninjured nerve of the same animals. These libraries were screened by differential hybridization and several transcriptionally repressed and induced sequences were isolated. Out of 2,000 cDNA clones screened from the distal library, 11 sequences were found to be induced in the distal nerve segment. This set of induced cDNAs included the rat homolog of vimentin, 28 S and 18 S ribosomal RNA species, and two novel sequences. Of 5,000 screened colonies of the contralateral library, 30 colonies contained sequences that were repressed in the distal segment after nerve crush. They were identified as myelin basic protein, myelin P0, α‐globin, cytochrome oxidase subunit 1, creatine kinase (muscle type, M) and collagen type I. In addition, five novel sequences were found that were dramatically repressed after sciatic nerve crush. Representative clones were tested by northern blot analysis to study their time course of transcriptional regulation during nerve regeneration. The observed patterns suggest that the regeneration phenomenon shows complex gene regulation in which the nonneuronal cells of the distal segment play an important role. Further characterization of the isolated regulated known and unknown sequences will increase our understanding of the molecular events associated with neuronal regeneration.
Glial growth is regulated by agonists selective for multiple opioid receptor types in vitro
Authors: Stiene‐Martin A. , Hauser K. F.
Published: January 2005

To determine whether one or more opioid receptor types might be preferentially involved in gliogenesis, primary mixed glial cultures derived from mouse ce‐rebra were continuously treated with varying concentrations of opioid agonists selective for mu (μ), delta DAGO ([D‐Ala2, MePhe4, Gly(ol) 5] enkephalin), (δ), i.e., DPDPE ([D‐PEN2, D‐PEN5]enkephalin), kappa (κ), i.e., U69,593, opioid receptor types. In addition, a group of cultures was treated with [Met5]‐enkephalin, an agonist for δ opioid receptors as well as putative zeta (ζ) opioid receptors. Opioid‐depen‐dent changes in growth were assessed by examining alterations in (1) the number of cells in mixed glial cultures at 3, .6, and 8 days in. vitro (DIV), (2) [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) as‐trocytes at 6 DIV, and (3) the area and form factor of GFAP‐immunoreactive, flat (type 1) astrocytes‐ DPDPE at 10−8 or 10−10 as M significant M enkephalin at 10−6, 10−8, 10−10 reduced the total number of glial cells in culture; but this effect was not observed with DAGO or U69, 593 (both at 10−6, 10−8 10−10) Equimolar or trations (i.e.), 10−6 M of [Met5]enkephalin U69,593, but not DPDPE or DAGO, suppressed the rate of [3H]thymidine incorporation by GFAP‐immunoreactive, flat (type 1) astrocytes. DAGO had no effect on growth, although in previous studies morphine was found to inhibit glial numbers and astro cyte DNA synthesis. [Met5]enkephalin (10−6 the only agonist to significantly influence astrocyte area. Collectively, these results indicate that δ (and perhaps μ) opioid receptor agonists reduce the total number of cells in mixed glial cultures; while [Met15]enkephalin‐responsive and perhaps κ‐respon‐sive) opioid receptors mediate DNA synthesis in astrocytes. This implies that δ opioid receptors, as well as [Met5]enkephalm, sensitive, non‐δ pioid receptors, mediate opioid‐dependent regulation of astrocyte and astrocyte progenitor growth. These data support the concept that opioid‐dependent changes in central nervous system growth are the result of endogenous opioid peptides acting through multiple opioid receptor types.
Some neural intermediate filaments contain both peripherin and the neurofilament proteins
Authors: Parysek L. M. , McReynolds M. A. , Goldman R. D. , Ley C. A.
Published: January 2005

Mammalian neurons and neuron‐like cultured cells express the neural intermediate filament (IF) proteins neurofilament (NF)‐L, NF‐M, NF‐H, and peripherin. To determine whether these proteins are found within the same 10‐nm filament, light and electron microscope immunocytochemistry using peripherin and NF‐specific antibodies was performed on PC12 cells, nervous tissue, and isolated neural filaments from the cauda equina. Double‐label immunofluorescence showed that peripherin and NF‐L, ‐M, and ‐H were found in identical filamentous patterns in interphase and mitotic PC12 cells. Furthermore, expression of mutant peripherin in PC12 cells disrupted not only the peripherin network but also NF‐containing filaments. Immunoelectron microscopy of PC12 cell cytoskeletons showed that peripherin and NF subunit proteins were found in the same filament. In situ, in the sciatic nerve, peripherin/NF‐L or peripherin/NF‐M/‐H double‐label immunofluorescence illustrates at least three types of nerve fibers: those containing NF only, those labeled predominantly for peripherin, and fibers in which peripherin and NF subunits were colocalized. Immunoelectron microscopy of filaments isolated from nerve roots comprising the sciatic nerve also showed the same three labeling patterns seen by light microscopy. Some neural IF appear to contain predominantly NF proteins or peripherin, but in others, both proteins are found within the same IF.
Maturation and aging of the axonal cytoskeleton: Biochemical analysis of transported tubulin
Authors: Tashiro T. , Komiya Y.
Published: January 2005

Changes in solubility and axonal transport of tubulin during maturation and aging have been investigated using sciatic motor fibers of rats at 4, 7, 14, 30, and 80 weeks of age. One to six weeks after injection of L‐[35S]methionine into the spinal cord, labeled cytoskeletal proteins in consecutive segments of the sciatic nerve and the ventral roots were fractionated into soluble and insoluble forms by extraction in 1 % Triton at low temperature. In 4‐week‐old rats, the two forms of tubulin were transported coordinately in a single wave with the average rate of 2 mm/day. At 7 weeks of age, two components in tubulin transport were observed to develop, possibly reflecting the maturation of the axonal cytoskeleton. The slower main component (1.5 mm/day) contained most of the insoluble form together with the neurofilament proteins and the faster component (3 mm/day) was enriched in the soluble form. Though significantly different in composition, the two components correspond to slow component a (SCa) and slow component b (SCb) originally defined in the optic system. A progressive decrease in transport rates of both SCa and SCb was observed with rats at 14, 30, and 80 weeks of age. In addition, there was a large decrease in the proportion of insoluble tubulin during the course of transport in animals older than 30 weeks. This loss of the insoluble form seems to be accounted for partly by the proteolytic degradation of the severely retarded SCa proteins. Changes in axonal transport of tubulin may thus reflect age‐related changes in dynamics and turnover of the axonal cytoskeleton.
Identification of cytoskeletal, focal adhesion, and cell adhesion proteins in growth cone particles isolated from developing chick brain
Authors: Cypher C. , Letourneau P. C.
Published: January 2005

Growth cones are intimately involved in determining the direction and extent of neurite elongation during development They are able to monitor their environment and respond to it by undergoing directed motility. We have isolated a fraction enriched in growth cone particles from embryonic chick brain. Assayed by immunoblots, this fraction is enriched in GAP‐43, and contains the cytoskeletal proteins actin, myosin II, neurofilament protein, tubulin, kinesin, and dynamin. All of the major components of focal adhesions are also present: alpha‐actinin, vinculin, talin, and integrin. In addition to integrin, we also identify the cell adhesion molecules A‐CAM, L1, fibronectin, and laminin in these particles. This preparation of isolated growth cone particles may be a useful model system for studying growth cone adhesion and motility.
Compliance of hippocampal neurons to patterned substrate networks
Authors: Corey J. M. , Wheeler B. C. , Brewer G. J.
Published: January 2005

Neuronal growth can be controlled in vitro by plating cells at low density and by differential adhesion between the cell and substrate. Primary cultures of rat hippocampal neurons were grown in serum‐free culture on polylysine‐coated glass coverslips patterned by selective laser ablation so as to leave grids of polylysine with varying linewidths (3, 5, and 10 μM), intersection distance (80, 120, and 160 μM), and nodal (intersection) diameter (5, 10, and 20 μM). Not only did somae strongly prefer the unablated polylysine areas, but they also migrated to foci where the local area of unablated polylysine was higher. These loci were the nodes, as opposed to the narrow connecting paths, and larger nodes, as compared with smaller nodes. Maximum migration to nodes of 88% occurred for a combination of 5‐μM pathwidth, 20‐μm node diameter, and 80‐μM pathlength. Daily observations indicated active migration to larger adhesive areas, which explains the differential compliance.
Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic CNS neurons: Comparison with basic fibroblast growth factor
Authors: Ferrari G. , Toffano G. , Skaper S. D.
Published: January 2005

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been described to exert neuronotrophic effects on central nervous system neurons in culture. To study the selectivity of trophic actions of these growth factors, neurotransmitter‐identified populations of embryonic rat mesencephalon were used. At 20 days in vitro, EGF (3 ng/ml) promoted survival and neurite outgrowth from these neurons. The neuritogenic effect of bFGF (3 ng/ml) was, however, more robust. Quantitative analysis with the neurofilament monoclonal antibody RT97 and ELISA confirmed the differential response, bFGF being 2–2.5 times more effective at all concentrations tested (ED100:3–10 ng/ml for both EGF and bFGF). At 10 days in vitro, EGF displayed no trophic activity — even at 30 ng/ml. Treatment of mesencephalic cultures with EGF (3 ng/ml) for 20 days stimulated [3H]dopamine and [14C]GABA uptakes about 4‐fold. While bFGF (3 ng/ml) also stimulated GABA uptake some 4‐fold, dopamine uptake was increased almost 20‐fold. Thus, EGF is also capable of enhancing the transmitter traits of selected central neuronal populations; however, the actions of bFGF appear to preferentially address dopaminergic cells.
Effect of CNTF on low‐affinity NGF receptor expression by cultured neurons from different rat brain regions
Authors: Magal E. , Burnham P. , Varon S.
Published: January 2005

Our previous work indicated that in E14 embryonic rat spinal cord cultures ciliary neuronotrophic factor (CNTF) exerted (1) a survival‐promoting effect on motor neurons and on a large population of unidentified neurons, and (2) a regulatory role on the expression of ChAT and low affinity NGF receptor (LNGFR) in a population of small/medium‐sized neurons. In the present study, we examined the effect of CNTF on the expression of LNGFR in cultures of different regions from the E18 embryonic rat brain, namely cortex, septum, striatum, mesencephalon, hippocampus, brainstem, and cerebellum. The number of LNGFR‐positive neurons (stained with the 192‐IgG monoclonal antibody) was determined in untreated cultures and in cultures treated for 6 days (0–6) with human recombinant CNTF. To distinguish between effects on survival and on LNGFR expression, experiments were performed in which CNTF was administered only for the last 48 h of the culture (from days 4–6). LNGFR positive neurons were found in the cultures of all the regions examined. In each one of them, CNTF increased the number of LNGFR‐positive neurons by three‐ to fourfold after 6 days of treatment. In the striatum, septum, mesencephalon, and cerebellum, the effect of CNTF was shown to be on the regulation of LNGFR expression and not on survival. In cultures from the cortex, hippocampus and brainstem, a survival‐promoting role of CNTF could be demonstrated. The effect of CNTF was dose dependent, with half‐maximal effects (ED50) achieved at 2–4.5 TU/ml for all the brain regions. Maximal effects were reached at 100–250 TU/ml. From these results, we conclude that (1) there exists a wide spectrum of CNTF‐responsive neurons in the central nervous system, and (2) CNTF plays an important and widespread role in regulating the expression of the LNGFR in neurons.
Peripheral nerve injury down‐regulates CNTF expression in adult rat sciatic nerves
Authors: Rabinovsky E. D. , Smith G. M. , Browder D. P. , Shine H. D. , McManaman J. L.
Published: January 2005

Ciliary neurotrophic factor (CNTF) is a 200‐amino acid protein expressed in high concentrations by peripheral nerves and is thought to be important for the survival and regeneration of injured motoneurons (Lin et al., J Biol Chem 265:8942–8947, 1990). To better understand CNTFs role in nerve injury we have characterized the effects of crush injury on the expression of CNTF in adult rat sciatic nerves using specific antibody and RNA probes. Following a crush injury, both the protein and mRNA levels undergo pronounced decreases distal to the crush. These changes in CNTF expression were qualitatively distinct from changes in the expression of the low‐af‐finity NGF receptor (p75NGFR), which increase following crush. Thus, the changes in CNTF levels do not reflect an overall down‐regulation of mRNA during degeneration, and are inconsistent with the proposed role of CNTF in neuronal injury, since its levels are decreasing at the same time as the requirement for neurotrophic support is increasing.
Further evidence for the dynamic formation of transmitter quanta at the neuromuscular junction
Authors: Vautrin J. , Kriebel M. E. , Holsapple J.
Published: January 2005

Fatt and Katz (Nature 166:597–598, 1950; J Physiol 117:109–128, 1952) attributed miniature endplate potentials (MEPPs) to the action of a standard quantity of transmitter, the quantum (Del Castillo and Katz, J Physiol 124:560–573, 1954). Quantal packets of transmitter were proposed to be preformed (Del Castillo and Katz, In CNRS Paris (Ed): “Microphysiologie comparée des éléments excitables” 67:245–258, 1957) and stored in large numbers in the motor nerve terminal. Statistical analyses of intervals between MEPPs and numbers of quanta composing small endplate potentials indicated that quantal release was a random process and that release sites functioned independently of each other. With the discovery of synaptic vesicles it was proposed that each contained one quantum of transmitter. The quantal‐vesicular hypothesis (Del Castillo and Katz, as cited above) fails, however, to explain amplitude distributions of MEPPs that are skewed and/or that show multiple peaks (Kriebel et al., Brain Res Review 15:167–178, 1990). The drop formation process (Shaw, “The Dripping Faucet as a Model Chaotic System,” Santa Cruz, CA: Aerial Press, Inc., 1984) was shown to generate amplitude classes of drops that were similar to classes of MEPPs which suggested that rapid changes in quantal size and ratios of skew‐ to bell‐MEPPs could be explained with a simple dynamic process which determines quantal size at the moment of release (Kriebel et al., as cited above, 1990). Further similarities between miniature endplate currents (MEPCs) and the formation of drops are reported here. We found that rapid changes in MEPC amplitudes and time courses, which accompany an increase in frequency, mimic changes in drop sizes that accompany increases in flow rate. MEPC intervals have a minimum and their distributions are comparable to those of drop intervals. During an increased rate of transmitter release, MEPP amplitudes and intervals were positively correlated. The results suggest that spontaneously released transmitter “packets” are formed at the moment of release and that transmitter supply to the process that forms packets is continuous. © 1992 Wiley‐Liss, Inc.
Intracellular calcium levels regulate the actions of nerve growth factor on calcium uptake in PC12 cells
Authors: Kozak A. , Nikodijevic B. , Yavin E. , Guroff G.
Published: January 2005

The uptake of divalent cations and the intracellular concentration of calcium in PC12 cells were studied by flow cytometric analysis using the calcium‐sensitive dye, Fluo‐3, under a variety of conditions. In particular the actions of nerve growth factor were analyzed. The data show that nerve growth factor stimulates the uptake of divalent cations and increases the intracellular calcium levels of cells attached to collagen‐coated plates. The data further indicate that nerve growth factor‐dependent increases in the uptake of divalent cations become less pronounced as the intracellular concentration of calcium increases. Intracellular calcium levels increase upon detachment of the cells from the plates and also with increasing cell density. Studies on the uptake of 45calcium confirmed the influence of intracellular calcium levels on nerve growth factor‐stimulated calcium uptake. Thus, the effect of nerve growth factor on the uptake of divalent cations is dependent on the calcium levels in the cells, perhaps explaining why previous studies in this field have provided inconsistent results. © 1992 Wiley‐Liss, Inc.
Mechanisms of nerve growth factor mRNA regulation by interleukin‐1β in hippocampal cultures: Role of second messengers
Authors: Friedman W. J. , Altiok N. , Fredholm B. B. , Persson H.
Published: January 2005

Cytokines such as interleukin‐1, which are found in the brain after trauma, regulate expression of nerve growth factor (NGF) mRNA and protein in hippocampal cultures. We have investigated possible mechanisms by which Il‐1β regulates NGF in hippocampal cells. The induction of NGF mRNA by Il‐1β was blocked by a receptor antagonist indicating that this effect is receptor mediated. Il‐1β elicited a dramatic induction of c‐fos mRNA and a slight elevation of c‐jun mRNA in a time dependent manner which may allow for a role in the induction of NGF mRNA expression. We examined whether specific second messenger pathways were involved in mediating the action of Il‐1β in the hippocampus. Activation of cAMP with forskolin or treatment with 8‐Br‐cAMP had no effect on NGF mRNA levels. Moreover, exposure of hippocampal cultures to Il‐1β evoked no change in cAMP levels, indicating that this second messenger system played little or no role in the regulation of NGF expression by Il‐1β in these cells. Further, interleukin‐1 elicited no change in membrane inositol phosphate turnover, nor did it affect intracellular calcium levels. Treatment of cell cultures with the phorbol ester PMA elicited an increase in NGF mRNA, suggesting that activation of protein kinase C (PKC) may mediate NGF mRNA expression. However, prolonged treatment of cultures with PMA to desensitize PKC did not eliminate the Il‐1β induction of NGF mRNA. Il‐1β, therfore, did not appear to activate NGF expression via cAMP, Ca2+, or a PKC isoform that is downregulated by prolonged PMA treatment. However, a phosphorylation event may be involved in the signal transduction mechanism, as treatment with okadaic acid to inhibit protein phosphatase 2a potentiated the induction of NGF mRNA by Il‐1β. The results presented indicate that Il‐1β acts via its receptor in induce a rise in NGF expression. Identification of the specific second messenger pathway has remained elusive; however, a phosphorylation event appears to be intermediary. Moreover, the induction of c‐fos and c‐jun may represent a final common path in activation of NGF gene expression by different signals such as Il‐1β and PMA. © 1992 Wiley‐Liss, Inc.
Cell cycle and proliferation dynamics of adult rat oligodendrocytes
Authors: Vick R. S. , Collins J. M. , Devries G. H.
Published: January 2005

Adult oligodendrocytes (OLGs) have been shown to be mitotically responsive when co‐cultured with dorsal root ganglion. We have investigated the population dynamics of the OLG proliferative response, including the maximum percent of cells which will proliferate and the cell cycle duration time of the adult rat OLG. Adult OLGs were stimulated with pituitary extract and either continuously labeled or pulse labeled with bromodeoxyuridine (BrdU). The labeled cells were stained with a fluorescent anti‐BrdU monoclonal antibody, and fluorescence measurements were made with a multiparameter flow cytometer. A maximum number of 53% of the adult OLGs entered the cell division cycle after 9 days. The cell division cycle duration time (Tc) was determined to be approximately 35.4 hr, and the durations of G1, S, and G2M found to be 12.1, 8.5, and 5.8 hr, respectively. A scenario was generated for adult OLG proliferation in which 2.2% of the initial population is stimulated to enter the cell division cycle in the first 24 hr. This initial population divides and continues in the cell division cycle. Another 2.2% of the initial population enters the cell division cycle during the second 24 hr period, divides, and continues in the cell division cycle. This entry of 2.2% of the cells into the proliferative phase continues until 53% of the initial population has left a quiescent state and entered the cell cycle. © 1992 Wiley‐Liss, Inc.
DM20 mRNA splice product of the myelin proteolipid protein gene is expressed in the murine heart
Authors: Campagnoni C. W. , Garbay B. , Micevych P. , Pribyl T. , Kampf K. , Handley V. W. , Campagnoni A. T.
Published: January 2005

Northern blot analysis of poly A(+) RNA isolated from mouse heart revealed the expression of 3.3 and 2.4 kb mRNAs that hybridized with a cDNA for the mouse proteolipid protein (PLP). In order to examine the relationship of these RNAs to the myelin PLP/DM20 mRNAs, a mouse heart cDNA library was prepared and screened with a mouse PLP cDNA. A cDNA was isolated, sequenced, and found to encode the DM20 variant of PLP. Polymerase chain reaction (PCR) analysis of heart cDNA with three sets of primers confirmed the presence of DM20 mRNA in mouse heart and indicated that it is the major splice product of the PLP gene expressed in that tissue. In situ hybridization localized the expression of the DM20 mRNA to the myocardial cells. Northern blot analysis indicated that expression of the DM20 mRNA is developmentally regulated in the murine heart, increasing significantly in concentration after 12 days postpartum. Northern analysis also revealed the expression of the DM20 mRNA in the hearts of the jimpy and quaking mutants. These results indicate that the PLP gene is expressed in tissues other than brain and support the concept that products of the PLP gene may have some biological role other than as structural components of myelin. © 1992 Wiley‐Liss, Inc.
Incorporation of 15N from L‐leucine into isoleucine by rat brain cerebral cortex slices
Authors: Gheorvassaki E. , Papadakis D. , Alikaridis F. , Kephalas T. , Larsen C. , Palaiologos G.
Published: January 2005

The fate of leucine nitrogen in the central nervous system was investigated by incubating rat cerebral cortex slices in the presence of 0.5 mM each of L‐[15N]‐leucine, L‐isoleucine, and L‐valine. Analysis of the slices and incubation media for free amino acids by gas chromatography‐mass spectrometry revealed that the 15N from leucine is incorporated into isoleucine only. No 15N was detected in valine or any other amino acid. These results suggest that leucine, valine, and their corresponding aminotransferases may be compartmentalized in brain cerebral cortex. © Wiley‐Liss, Inc.
Oligodendrocyte differentiation and progenitor cell proliferation are independently regulated by cyclic AMP
Authors: Raible D. W. , McMorris F. Arthur
Published: January 2005

Oligodendrocytes, the glial cells specialized to synthesize myelin in the central nervous system, differentiate in primary rat brain cell cultures on a schedule similar to that observed in vivo. The schedule of oligodendrocyte differentiation and the rate of oligodendroglial progenitor cell proliferation in vitro are both modulated by 3′,5′‐cyclic AMP (cAMP). A 24‐hour exposure to 1 mM N6,2′O‐dibutyryladenosine 3′,5′‐cyclic monophosphate (dbcAMP) induced a wave of oligodendrocyte differentiation but inhibited proliferation of oligodendroglial progenitors, and reduced by 30‐fold the proliferation of progenitors in response to platelet‐derived growth factor (PDGF). When cells were grown in the presence of maximally stimulating concentrations of PDGF, the inhibitory effect of cAMP on progenitor cell proliferation was abolished while the stimulatory effect of cAMP on oligodendrocyte differentiation remained, demonstrating that these two cAMP‐regulated events are independent. © 1993 Wiley‐Liss, Inc.
Cytotoxic actions and effects on intracellular Ca2+ and cGMP concentrations of sulphur‐containing excitatory amino acids in cultured cerebral cortical neurons
Authors: Frandsen A. , Schousboe A. , Griffiths R.
Published: January 2005

Effects of the sulphur‐containing acidic amino acids (SAAs) cysteic acid (CA), homocysteic acid (HCA), cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), and S‐sulphocysteine (SC) on intracellular concentrations of Ca2+ ([Ca2+]i) and cGMP ([cGMP]i) as well as their cytotoxic actions were investigated in cultured cerebral cortical neurons. The glutamate receptor subtype selective antagonists APV (D‐(−)‐2‐amino‐5‐phosphonopentanoate) acting on N‐methyl‐D‐aspartate (NMDA) receptors and DNQX (6,7‐dinitroquinoxaline‐2,3‐dione) acting on non‐NMDA receptors were employed to obtain information about the involvement of glutamate receptor subtypes in these actions of the SAAs. It was found that all SAAs exerted a cytotoxic action on the neurons. The ED50 values for CSA, CA, HCSA, and HCA were around 30 to 50 μM and that for SC was about 150 μM. The glutamate transport blocker L‐aspartate‐β‐hydroxamate increased the efficacy of CSA and CA but had no effect on the cytotoxic actions of the remaining SAAs. In case of CA, HCA, and SC the cytotoxicity could be prevented by APV alone and for HCSA, DNQX could block the toxic action. DNQX reduced the toxicity of HCA somewhat but the presence of APV was required for complete protection. CSA toxicity could only be blocked by the combination of APV and DNQX. All SAAs induced an increase in [cGMP]i and [Ca2+]i and with regard to [Ca2+]i SC was the most potent and CA the least potent SAA. The effect of all SAAs on [cGMP]i could be blocked by APV alone whereas DNQX had no effect except in the case of HCSA where the response was blocked completely and HCA where the response was inhibited by 75%. The SAA‐induced increase in [Ca2+]i could in all cases be significantly reduced by 0.6 mM Mg2+ and in the presence of Mg2+, APV dose dependently blocked the remaining SAA induced increase in [Ca2+]i completely. Under these conditions DNQX was also found to block the SAA‐induced increase in [Ca2+]i dose dependently. In the absence of Mg2+, DNQX (25 μM) inhibited the response of the SAAs only by 65–75%. Under these conditions all SAA responses except that to SC could be fully antagonized by 300 μM APV. The SC‐induced increase in [Ca2+]i was inhibited by 60% by APV. The results show that no simple correlation exists between SAA‐induced cytotoxicity and their ability to increase intracellular levels of Ca2+ and cGMP. However, when both NMDA and non‐NMDA receptors were antagonized no toxicity or changes in calcium or cGMP were observed. © 1993 Wiley‐Liss, Inc.
Selective modulation of cholinergic properties in cultures of avian embryonic sympathetic ganglia
Authors: Smith J. , Vyas S. , Garcia‐Arraras J. E.
Published: January 2005

We have studied the expression of catecholaminergic and cholinergic phenotypes in sympathetic ganglia removed from 7‐ to 10‐day‐old quail embryos and grown in vitro under different conditions. Quantitative data were obtained by measuring the conversion of (3H) tyrosine and (3H) choline to catecholamines (CA) and acetylcholine (ACh), respectively. In explant cultures, large amounts of both neurotransmitters were synthesized from the onset, but CA generally predominated, the molar ratios of CA:ACh being, on average, of the order of 2:1. If the ganglia were dissociated before plating, there was a selective increase in ACh synthesis (three‐ to fivefold) such that the CA:ACh ratio fell strikingly. The early expression of the cholinergic phenotype appears to be species‐specific in that, under identical conditions, dissociated cell cultures of newborn mouse superior cervical ganglia were overwhelmingly catecholaminergic (CA:ACh ratio of approximately 40:1) and ACh synthesis was only just detectable. Addition of veratridine (1.5 μM) either to explant or to dissociated cell cultures of embryonic quail sympathetic ganglia barely altered CA‐synthesizing ability; in contrast, ACh synthesis and accumulation were stimulated about threefold. This effect, which we found to correspond to a quantitatively similar increase in the activity of choline acetyltransferase (ChAT), was completely blocked by tetrodotoxin, indicating that it was due to Na+‐dependent depolarization. A preferential stimulation of ACh production was also observed when the concentration of K+ was raised to 20 mM. Veratridine treatment of cultures of presumptive sympathoblasts, in the form of sclerotome‐associated neural crest cells, had identical effects. Our results reveal the quantitative importance of ACh‐related properties in avian sympathetic ganglia from the earliest stages of their development and suggest that depolarization may be one of the factors selectively enhancing expression of the cholinergic phenotype during ontogeny. In these respects, the neurochemical differentiation of sympathetic neurons unfolds according to dissimilar scenarios in birds and mammals. © 1993 Wiley‐Liss, Inc.