bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose transport in primary cultured neurons
Authors: Heidenreich K. A. , Gilmore P. R. , Garvey W. T.
Published: January 2005

In this study, we have examined glucose uptake and its regulation by insulin in primary cultured neurons. Glucose transport was assessed by measuring the initial rate of uptake of 3H‐2‐deoxyglucose, a glucose analog that is transported and phosphorylated but not further metabolized. The uptake of 2‐deoxyglucose was saturable; measurements of the intracellular concentration of 2‐deoxyglucose and 2‐deoxyglucose‐6‐phosphate revealed that hexokinase activity rather than membrane transport is the rate‐limiting step for glucose uptake. Insulin had no effect on 2‐deoxyglucose uptake at low (0.2 mM) or high (20 mM) concentrations of substrate. The order of potency of other hexoses to competitively inhibit the accumulation of 2‐deoxyglucose was D‐glucose (0.2 mM) = D‐mannose (0.2 mM) > 30‐methylglucose (9 mM) > D‐galactose (90 mM). Cytochalasin B was a potent inhibitor of 2‐deoxyglucose uptake (IC50 = 500 nM) and phloretin was more potent than ploridzin in inhibiting uptake. The structure of glucose transporters was examined by photoaffinity labeling using 3H‐cytochalasin B and by immunologic detection using antibodies raised against the human erythrocyte transporter. 3H‐cytochalasin B labeled two proteins of 55 kDa and 43 kDa and the antibody recognized primarily a 43 kDa protein. The subcellular distribution of glucose transporters, estimated by measuring the number of specific cytochalasin B binding sites in subfractions of neuronal homogenates, showed 3.62 pmol/mg protein in the 11,000g pellet and 1.34 pmol/mg protein in the 200,000g pellet.

In conclusion: (1)Neuronal glucose transport is not acutely regulated by insulin. (2) The kinetics of 2‐deoxyglucose uptake into neurons are determined largely by hexokinase activity rather than membrane transport. (3) The apparent molecular weight of neuronal glucose transporters is similar to transporters in other tissues. (4) The number of glucose transporters per milligram of protein is relatively low in neurons compared to other tissues.
Role for the oligodendrocyte cytoskeleton in myelination
Authors: Wilson R. , Brophy P. J.
Published: January 2005

Enriched cultures of rat brain oligodendrocytes were extracted with a buffer that separated the cells into a Triton X‐100—soluble fraction and an insoluble cytoskeleton (CSK) residue. The buffer was optimised so that intact microtubules were preserved in the CSK residue. The partition of four myelin proteins between the soluble and the CSK fractions was determined by immunoblotting and immunofluorescence. Immunoblotting showed that two integral membrane proteins of myelin, the proteolipid protein (PLP) and the DM‐20 protein, were completely extracted under these conditions. By contrast, a substantial amount of myelin basic protein (MBP) and to a lesser extent 2,3‐cyclic nucleotide‐3‐phosphohydrolase (CNP) remained associated with the CSK residue. The association of these proteins with the CSK was confirmed by immunofluorescence. A remarkable difference in the distribution of microfilaments and microtubules was observed in oligodendrocytes. Immature cells possessed many fine processes that were rich in microfilaments. The cell body of these oligodendrocytes was devoid of microfilaments but did contain microtubules. Furthermore, a close association between CNP and microfilaments and between MBP and microtubules was revealed after detergent lysis. The strong interaction between CNP and filamentous actin was underlined by their concomitant disappearance from the extremeties of the cell at a later stage of development when extensive membrane sheets had formed. Mature cells had fewer, thicker processes than younger cells and their processes contained microtubules, not microfilaments. MBP was present throughout the thick processes and the membrane sheets. These observations suggest roles for CNP and MBP at distinct stages of myelin process formation and support a directive role for the oligodendrocyte's CSK in the formation of myelin.
Characterization of β‐adrenoceptors in the syrian hamster harderian gland: Sexual differences and effects of either castration of superior cervical ganglionectomy
Authors: Pangerl A. , Pangerl B. , Buzzell G. R. , Jones D. J. , Reiter R. J.
Published: January 2005

Scatchard analysis of saturation isotherms of [125I]‐iodopindolol was used to characterize β‐adrenoceptor density (Bmax) and affinity constant (Kd) in female and male hamster Harderian glands. Single‐point experiments were also completed in intact females, intact males, and castrated or superior cervical ganglionectomized males. Scatchard analysis described a single population of binding sites with a Bmax of 292.2 ± 45.1 fmol/mg protein (X ± SEM, n = 6) in females and 18.2 ± 3.0 fmol/mg protein (n = 6, P < 0.001) in males. The affinity also varied significantly (P < 0.05) with a Kd of 1.08 ± 0.18 versus 0.26 + 0.05 nM (n = 6) in the Harderian gland of females and males, respectively. Single‐point [125I]‐IPIN (400 pM) binding values in females were 67.3 ± 4.0, in intact males were 12.8 ± 3.2, and in castrated males were 31.2 ± 4.2 fmol/mg protein (n = 7–9). Superior cervical ganglionectomy induced no significant changes in receptor binding. The results indicate pronounced sexual differences in the density and affinity of β‐adrenoceptors in the hamster Harderian gland, which may be sex hormone dependent.
Intracerebroventricular administration of bombesin inhibits biliary and gastric secretion in the rat
Authors: Yao C. Z. , Maclellan D. G. , Thompson J. C.
Published: January 2005

The effect of intracerebroventricular (ICV) bombesin on bile and gastric acid secretion was examined in the rat. ICV bombesin (10 μg in 10 μl) inhibited basal biliary volume by 27% and bile bicarbonate by 52% of control values, while gastric acid secretion was decreased by 75%. These results provide evidence for central mechanisms for the control of gastrointestinal function.
Na, K‐ATPase: Comparison of the cellular localization of α‐subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney
Authors: Hieber V. , Siegel G. J. , Desmond T. , Lee‐Hwa Liu J. , Ernst S. A.
Published: January 2005

A clone encoding mouse brain Na, K‐ATPase α‐subunit was isolated from a mouse brain lambda gt11 cDNA library by using antisera to mouse and bovine brain α‐subunit A comparison of the nucleotide sequence of this clone with published sequences of rat brain α‐subunit isoform clones showed it to be most similar to rat brain α1. An RNA antisense probe prepared from the cDNA insert of the mouse clone detected a single mRNA of approximately 4.5 kb in Northern blots of mouse brain and kidney RNAs. This probe hybridized only to an α1‐cDNA insert from rat brain under high stringency conditions on Northern blots. The RNA antisense probe was used for in situ hybridization to sections of mouse kidney, cerebellum, and retina, and the cellular distribution of α‐subunit mRNA (α‐mRNA) was compared with that of α‐subunit polypeptide (α‐subunit) detected by immunofluorescence in similar sections. In kidney, α‐mRNA distribution closely paralleled that of the polypeptide with abundant expression in ascending thick limbs and cortical distal tubules and weaker labeling in cortical proximal tubules. The co‐distribution of α‐mRNA and polypeptide in kidney where Na, K‐ATPase localization is well established is consistent with the specificity of these probes. In the retina, prominent labeling with both probes was seen in photoreceptor inner segments, inner nuclear layer, and ganglion cell bodies. Plexiform layers and optic fibers expressed abundant α‐subunit but little mRNA. Light labeling for both was seen in the outer nuclear layer. In cerebellum, α‐mRNA and α‐subunit were associated with soma of granule cells, basket cells, and stellate cells. Glomeruli and basket terminals contained abundant α‐subunit but exhibited little reactivity with the riboprobe. In Purkinje cell bodies, in contrast, the antibody used to identify the cDNA clone did not resolve significant polypeptide in the somal plasmalemma despite abundant somal mRNA expression. Comparison of distribution of the two probes in cerebellum and retina indicates that message accumulation is primarily in cell bodies, while α‐subunit epitopes may be co‐expressed in cell bodies and/or transported to distant sites in cell‐specific patterns.
Expression of pyruvate kinase in astrocytes induced to differentiate in vitro
Authors: Ngo J. L. , Ibsen K. H.
Published: January 2005

Astrocytes maintained in a chemically defined media undergo differentiation and a parallel increase in pyruvate kinase specific activity. These changes are accompanied by a shift in the isoelectrofocusing pattern, but not by expression of pyruvate kinase M4 the characteristic adult rat brain isozyme. Thus, this chemically defined media lacks a substance required to induce pyruvate kinase M synthesis and this function can be uncoupled from other aspects of cellular differentiation. The uncoupling of pyruvate kinase maturation from cellular differentiation and the observation of only a single, 2.3 kilobase, pyruvate kinase mRNA molecule at different stages of the postnatal development of rat brain support the concept that the K‐ to M‐isoform transformation is a posttranscriptional event.

The effect of the individual components of this chemically defined medium on pyruvate kinase specific activity was studied by eliminating one component at a time. The increase in activity was found to be completely dependent upon fibroblastic growth factor and prostaglandin F2α and was partially dependent on the simultaneous presence of insulin.
Development and regional expression of chicken neuroleukin (glucose‐6‐phosphate isomerase) messenger RNA
Authors: Hallböök F. , Persson H. , Barbany G. , Ebendal T.
Published: January 2005

Neuroleukin (NLK) is a protein identical with the glycolytic enzyme glucos‐6‐phosphate isomerase (GPI) that has been reported to support the survival of a subpopulation of neurons in embryonic dorsal root ganglia and spinal cord neurons in culture.

In this report we have studied the developmental expression of NLK mRNA in the chick embryo in order to evaluate its possible role as a neurotrophic factor. The chicken gene encoding NLK was isolated by cross‐hybridization to a mouse NLK cDNA clone. A DNA fragment from the chicken NLK gene with a 90% nucleotide sequence homology to mouse NLK cDNA encoding amino acids 310–355 was then used as a hybridization probe in a series of RNA‐blots. In the entire embryo NLK mRNA was found already at embryonic day 3.5 (E3.5) and the level of expression was significantly decreased between E3.5 and hatching. Roughly similar levels of NLK mRNA were found in all tissues of the E8 embryo analyzed, with the exception of the brain, which contained only low levels. When the developmental expression was analyzed in different tissues separately, NLK mRNA expression was found to decrease during development in the heart and bursa of Fabricius, whereas the level of mRNA in the brain showed a large increase shortly after hatching. The spinal cord and the pectoral and femoral muscles all showed high levels of NLK mRNA throughout development. In the adult chick, the highest levels of NLK mRNA were found in the muscle, brain, and kidney, where the NLK mRNA was estimated to account for approximately 0.1% of the total mRNA in these tissues. A widespread expression of NLK mRNA was observed in the adult brain with approximately similar levels in all brain regions tested. Similar results were also obtained when NLK mRNA expression was analyzed in adult rats. Our results show that developmental expression of the NLK gene is independently regulated in different tissues. The widespread and abundant expression of both the avian and rodent NLK gene is in accordance with its newly discovered identity as a glycolytic enzyme. Consequently, the developmental and adult pattern of NLK mRNA expression does not favour a specific trophic role for this protein in accordance with other known neurotrophic factors.
Effect of nerve growth factor on the elongation of neurites from axotomized rat embryonic septal‐basal forebrain neurons: An in vitro analysis
Authors: Schinstine M. , Cornbrooks C. J.
Published: January 2005

The administration of nerve growth factor (NGF) into the brain of a fornix‐fimbria lesioned rat can rescue many cholinergic, septal‐basal forebrain (SBF) neurons from imminent cell death. Unfortunately, it is nuclear if NGF can stimulate regenerative growth from axotomized, SBF neurons. In the present study, we used an in vitro model system to determine if NGF could affect neurite outgrowth from nonaxotomized and/or axotomized, embryonic SBF neurons. Axotomized neurons were obtained by severing the neuritic fields surrounding embryonic day (E) 15 SBF explants maintained in primary culture. Acetylcholinesterase (AChE) histochemistry was used to assess the effects of NGF on cholinergic neurites. We report that (1) neurite outgrowth on type I collagen from E15 SBF neurons in primary culture (nonaxotomized neurons) was not affected by NGF. (2) NGF enhanced the outgrowth (regeneration) of axotomized, SBF neurons on a collagen substratum; however, neurons had to be treated with NGF both before and after axotomy to stimulate regeneration effectively. Application of NGF either before or after axotomy did not enhance regenerative neurite outgrowth. (3) SBF neurons had to be axotomized for NGF to facilitate neurite outgrowth. This is supported by the observation that SBF explants, initially maintained in NGF‐supplemented medium in suspension culture, did not demonstrate enhanced neurite outgrowth in the presence of NGF when plated onto a substratum. (4) The regenerative growth of AChE‐negative, as well as AChE‐positive, neurites was facilitated by NGF treatment. In addition to data concerning neurite outgrowth, we also found that the NGF receptor, as recognized by the antibody 192‐IgG, expands its distribution as time in culture progresses; i.e., staining, originally confined to cell bodies and proximal processes within the explant, later included neurites that emanated from the explant. Thus, our results demonstrate that NGF can stimulate regenerative growth from axotomized, but not nonaxotomized, embryonic SBF neurons. We hypothesize that, given the appropriate substratum for axon elongation in vivo, NGF can stimulate the regeneration of SBF neurons in the injured adult brain.
Thiamine‐like molecules in the cerebellar cortex of the rat: Light and electron microscopic immunocytochemical investigation
Authors: Martínez‐Rodriguez R. , Díaz G. Arenas , Carnicero M. B.
Published: January 2005

Immunocytochemical methods were used for studying the location of thiamine‐like molecules in the cerebellar cortex of the rat. A positive reaction was observed in several synapses and in several dendrites. This reaction was associated with tubular formations and synaptic vesicles. A positive reaction was also found in several mitochondria and in glial cells. The possible role of thiamine‐like molecules in cerebellar cortex is discussed.
Increase in cathepsin D activity in rat brain in aging
Authors: Kenessey A. , Banay‐Schwartz M. , de Guzman T. , Lajtha A.
Published: January 2005

Cathepsin D‐like activity in homogenates of five brain areas of 3‐month‐old and 24‐month‐old Fischer 344 rats was measured. With hemoglobin as substrate at pH 3.2, more than 90% of the activity was inhibited by pepstatin. In each area studied, activity was more than twice as high in the old rat brain: 140–160% higher in the cortex, cerebellum, pons‐medulla, and striatum and 90–100% higher in the hippocampus and spinal cord. The greatly increased metabolic capacity in the absence of an increase in protein turnover may have a role in age‐related pathological degeneration in the brain.
Acetyl‐L‐carnatine reduces the age‐dependent loss of glucocorticoid receptors in the rat hippocampus: An autoradiographic study
Authors: Patacchioli F. R. , Amenta F. , Ramacci M. T. , Taglialatela G. , Maccari S. , Angelucci L.
Published: January 2005

Brain autoradiography in adrenalectomized rats injected with 3H‐corticosterone 2 hr before sacrifice was used to study the effect of aging and long‐term acetyl‐l‐carnitine treatment on the hippocampal glucocorticoid receptor.

Densitometric analysis of silver grains in individual nerve cells of the hippocampus showed that pyramidal neurones of the CA1 field and granular cells of the dentate gyrus are richest in 3H‐corticosterone binding sites, whereas pyramidal neurons of the CA3 field have the lowest number of binding sites.

There was a significant decline in the number of glucocorticoid receptors within the various hippocampal areas, both as the total number of 3H‐corticosterone binding sites and as the number per single pyramidal or granule neuron associated with aging and perhaps due to loss of adrenocorticoid‐competent neurons. The dentate gyrus and the CA1 region were mostly affected by the age‐dependent decrease in glucocorticoid receptors of the hippocampus.

Twenty‐eight‐month‐old rats, treated with acetyl‐l‐carnitine for 7 months, showed a significantly higher number of 3H‐corticosterone binding sites within the various hippocampal regions examined than did age‐matched controls. The CA1 and the dentate gyrus were the regions most susceptible to amelioration by acetyl‐l‐carnitine treatment.

These findings suggest a positive effect of acetyl‐l‐carnitine treatment on age‐related changes which occur in the hippocampus.
Role of the c‐myc and the N‐myc proto‐oncogenes in the immortalization of neural precursors
Authors: Bernard O. , Reid H. H. , Bartlett P. F.
Published: January 2005

To study the role of c‐myc and N‐myc in the immortalization of neural precursors, we infected neuroepithelial cells isolated from 10‐day‐old mouse embryos with a new retrovirus vector, pzen, harboring either the c‐myc or the N‐myc oncogene. The immortalized cell lines contain high levels of the virally expressed myc protein. The amount of myc proteins correlated with the capacity of the cells to differentiate spontaneously in vitro into neurons and glia; cell lines expressing high levels of myc protein can differentiate spontaneously while cell lines expressing low levels of the myc protein resemble epithelial cells. Addition of acidic or basic fibroblast growth factor enhanced differentiation of most cell lines. Some of the cell lines produced neurotrophic growth factors capable of supporting the growth of other cell lines at low density. There was no significant difference between cell lines immortalized with c‐myc or with N‐myc. Most of the immortalized cell lines generated from bipotential precursors are capable of differentiating into neurons and glia.
Magnocellular neurosecretory axon regeneration into rat intrahypothalamic optic nerve allografts
Authors: Dellmann H.‐D. , Lue L.‐F. , Bellin S. I. , Quassat M.
Published: January 2005

To test the working hypothesis that neurosecretory neuronal regeneration is largely dependent on microenvironmental conditions at the lesion site, intact or predegenerated optic nerves were allografted intrahypothalamically into the hypothalamo‐neurohypophysial tract. Neurosecretory axons regenerated consistently into all grafts. Early regeneration proceeded without glial cell association. At later stages of regeneration, however, neurosecretory axons were associated consistently with astrocytes that, within perivascular spaces, were surrounded by a basal lamina. Axons in contact with that basal lamina had the characteristics of terminals, suggesting functional recovery. It is postulated that it is the initial absence of a blood‐brain barrier at the graft site that provides a microenvironment similar to that in the neural lobe and that induces neurosecretory axon regeneration.
Phosphatidylinositol‐specific phospholipase C solubilized G2 acetylcholinesterase from plasma membranes of chromaffin cells
Authors: Prieto A. L. , Fuentes M. E. , Arqueros L. , Inestrosa N. C.
Published: January 2005

Using whole homogenates and defined subcellular fractions of bovine adrenal medulla, we investigated the properties of the dimeric G2 molecular form of acetylcholinesterase (AChE), its distribution, and the mode of attachment to chromaffin cells. Our studies indicate that a substantial fraction of the G2 form is specifically susceptible to solubilization by phosphatidylinositol‐specific phospholipase C (PIPLC) from subcellular fractions enriched with plasma membrane fragments. The results suggest that the G2 form of AChE is anchored in the plasma membrane to a glycolipid domain that contains phosphatidylinositol. Since a Ca+2‐dependent PIPLC has been previously described in chromaffin granules, it is possible that the adrenal AChE could be released by a system reminiscent of that involved in the case of the surface glycoprotein of Trypanosoma brucei.
Coated vesicles from developing and adult rat skeletal muscles contain multiple molecular forms of acetylcholinesterase
Authors: Hodges‐Savola C. A. , Gregory E. J. , Rummel S. A. , Fernandez H. L.
Published: January 2005

The main purpose of this work was to determine which of the multiple isoforms of acetylcholinesterase (AChE) are associated with clathrin‐coated vesicles (CVs) from developing and adult rat skeletal muscles. CV‐enriched preparations were obtained by subcellular fractionation/equilibrium sedimentation and further purified by immunoadsorption to anti‐clathrin IgG‐coated Staphylococcus aureus cells. Analysis of individual AChE isoforms by velocity sedimentation ultracentrifugation showed that (a) while both globular (G‐forms) and asymmetric (A‐forms) AChE were detected in all subcellular fractions evaluated, the CV‐enriched fraction contained a higher proportion of A‐forms (mainly the A12 species); (b) most of the AChE activity contained in such a CV fraction was recovered following immunoadsorption; (c) alkaline extraction conditions (pH 8.5) which depolymerize clathrin were necessary to detect a large proportion of A‐forms in both the CV‐enriched and immunoprecipitated preparations, while most of the G‐forms (especially G1 + G2 AChE) were detected following extraction at pH 6.8; and (d) comparison of AChE isoform profiles from neonate and adult muscle CV‐enriched fractions showed a greater concentration of A‐forms in the former. These data suggest that considerable amounts of A12 and, to a lesser extent, G4 AChE are sequestered within muscle CVs which may be destined for the plasmalemma. Our findings also indicate that the relative proportions of AChE isoenzymes in rat muscle CVs vary according to the extent of muscle development and lend support to the contention that CVs participate in the externalization of functionally important AChE isoenzymes.
Adenosine metabolism in neurons and astrocytes in primary cultures
Authors: Matz H. , Hertz L.
Published: January 2005

Metabolic fate of [8‐14C]adenosine was studied in primary cultures of either astrocytes or neurons from the mouse brain. In astrocytes the main metabolic route was the formation of nucleotides. Thus, synthesis of adenosine triphosphate (ATP) amounted to about 0.2 nmol × min−1 × mg−1 protein. The deamination occurred less rapidly. The total rate of formation of inosine was difficult to establish because a considerable amount of labeled inosine accumulated in the medium. The initial incorporation of radioactivity into inosine in the medium was extremely rapid, probably because of the action of an ectoenzyme. However, the labeling of inosine in the medium also continued to increase slowly throughout the incubation, maybe as a result of release of intracellularly formed inosine. The total inosine formation rate during the incubation amounted to at most 0.1 nmol × min−1 × mg−1. Hypoxanthine was formed at a corresponding rate but was released to a lesser extent. In neurons much less label was incorporated into ATP. The major metabolite was inosine, formed intracellularly at a rate of 0.2 nmol × min−1 × mg−1. In addition, there was an immediate rapid labeling of inosine (and to a lesser extent hypoxanthine) in the medium, again suggesting the action of an ectoenzyme. Neither neurons nor astrocytes released a measurable amount of nucleotides to the medium.

The cellular differences in adenosine metabolism are probably of relevance for the interpretation of adenosine metabolism in brain in situ. The ectoenzyme may be of importance for rapid termination of the neuromodulator activity of adenosine, and the rapid nucleotide formation in astrocytes is in agreement with a high metabolic activity of these cells.
Uptake and release of glycine in cerebellar granule cells and astrocytes in primary culture: Potassium‐stimulated release from granule cells is calcium‐dependent
Authors: Holopainen I. , Kontro P.
Published: January 2005

The properties of [3H]glycine uptake and release were studied with cerebellar granule cells, 7–9 days in vitro, (DIV) and astrocytes, 14–15 DIV, in primary cultures. The uptake of glycine in both cell types consisted of a saturable high‐affinity transport and nonsaturable diffusion. The transport constant (Km) and maximal velocity (V) were significantly higher in granule cells than in astrocytes. Uptake was strictly Na+‐dependent and also markedly diminished in low Cl medium. The specificity of the uptake was similar in both cell types. The spontaneous release of glycine from granule cells and astrocytes was fast. Homoexchange with extracellularly added glycine in granule cells suggests that the efflux is at least partly mediated via membrane transport sites in these cells. Kainate stimulated the release more effectively in neurons than in glial cells, the effect apparently being mediated by specific kainate‐sensitive receptors in both cell types. The release was enhanced by veratridine and by depolarization of cell membranes by high K (50 mM) in both neurons and astrocytes. The potassium‐stimulated release was partially Ca‐dependent in neurons but Ca‐independent in glial cells. The results suggest a functional role for glycine in both cerebellar astrocytes and glutamatergic granule cells.
EGF‐induced neuritogenesis and correlated synthesis of plasma membrane gangliosides in cultured embryonic chick CNS neurons
Authors: Rosenberg A. , Noble E. P.
Published: January 2005

Epidermal growth factor (EGF), over a low range of concentrations (165–825 pM), induced neuritogenesis in post‐mitotic chick CNS precursor neurons cultured in a serum‐free medium, without the addition of other growth factors. Antibody to EGF blocks the neurite‐promoting activity of EGF. Similarl, neuritogenesis of cultured chick CNS neurons in medium supplemented with 20% fetal bovine serum is blocked by antibody to EGF, even though serum may contain other neuronotrophic bioactive proteins and steroids.

Quantitatively, the only major gangliosides of the undifferentiated post‐mitotic neurons are GD3 and GD2.1 GD3 as well as its biosynthetic precursor, GM3, undergo active biosynthesis in serum‐free medium as evidenced by their vigorous labeling by radioactive galactose supplied in the culture medium. When the undifferentiated neurons in serum‐free medium are exposed to EGF, the ensuing generation of neurite plasma membrane coincides with initiation of biosyn thesis of the sialosyl gangliotetraosyl ceramide species of gangliosides (GD1A, GD1B, GT1B, GQ1B). Antibody to EGF simultaneously inhibits biosynthesis of these gangliosides as well as inhibition of neuritogenesis.

These findings indicate that EGF may be a primary neurite‐inducing growth factor for post‐mitotic embryonic CNS neurons and that gangliosides, particularly those of the sialosyl gangliotetraosyl ceramide species, characterize the plasma membrane of CNS neurons during neuritogenesis.
T lymphocytes can recognize determinants unique to neuropeptides of guinea pig myelin basic protein containing a single D‐isomer amino acid substitution
Authors: Levy R. B. , Miller S. , Garcia N. M. , Jones M. , Fisher G. H. , Man E. H.
Published: January 2005

The present studies were undertaken to examine how the substitution of racemized forms of selected amino acids in synthetic peptides of guinea pig myelin basic protein (GPMBP) would after the host's immunological ability to recognize such molecules. Using peptides from the 69–84 sequence of GPMBP containing a D‐serine at position 70 or 75 (69–84[D‐ser70 or D‐ser75]) or D‐asparate at position 82 (69–84[D‐asp82]), the findings demonstrated that the position of the diastereomer substitution on tthese neuropeptides was critical with respect to the ability of the immune system to recognize the molecule. Thus substitution of D‐asp at position 82 or D‐ser at position 75 abrogated the ability of these peptides to induce experimental autoimmune encephalitis and proliferation of host T cells. In contrast, a peptide containing a D‐ser70 residue was capable of inducing clinical disease in rats, as well as stimulating T lymphocytes from 69–84‐(D‐ser70)‐injected animals. Moreover, although this D‐peptide was shown to share at least determinant(s)‐stimulated cell lines demonstrated that some epitope(s) unique to this molecule could stimulate CD4+ syngeneic T cells.
Effects of MK‐801 on glutamate‐induced swelling of astrocytes in primary cell culture
Authors: Chan P. H. , Chu L. , Chen S.
Published: January 2005

The effects of glutamate and its agonists and antagonists on the swelling of primary astrocytes were studied. Glutamate (Glu), aspartate (Asp), homocysteate (HCA), and quisqualate (Quis) at 1 mM concentration caused a significant increase in astrocytic swelling as measured by the 3‐0‐methyl‐[14C]‐glucose, whereas kainate (KA), N‐methyl‐D‐aspartate (NMDA), and receptor antagonists 2‐amino‐5‐phosphonovaleric acid (APV), 2‐amino‐7‐phosphonohepatanoic acid (APH), and kynurenic acid (Kynu) were not effective. This glial swelling was time‐dependent since 1‐hr or greater incubations with Glu or its agonists were needed to produce such an effect. Preincubation of glutamate or NMDA receptor anatogonists including Kynu, APH, and APV failed to ameliorate the Glu effects. However, MK‐801, a noncompetitive NMDA antagonist, when added to the Glu‐incubated astrocytes significantly reduced Glu‐induced astrocytic swelling. MK‐801 was also effective in reducing the astrocytic swelling induced by agonists including Asp, Quis, and HCA, suggesting that those agonists may share similar mechanisms of Glu in inducing astrocytic swelling. Since the cultured astrocytes lack the NMDA receptors, our data suggest that the observed beneficial effects of MK‐801 on excitotoxin‐induced swelling of astrocytes may be mediated by mechanisms other than NMDA receptors.
Modification of serotonergic innervation of the rat spinal cord dorsal horn after neonatal capsaicin treatment
Authors: Marlier L. , Rajaofetra N. , Poulat P. , Privat A.
Published: January 2005

Newborn rats were treated with Capsaicin. Seven weeks later, calcitonin gene‐related peptide, substance P, and serotonin were detected immunocytochemically in the dorsal horn of the spinal cord. Peptide immunostaining, presumably located in primary afferents, was reduced in intensity, whereas the pattern of serotonin innervation was modified, suggesting a secondary reorganization due to the lesion of afferents. The possible mechanisms of this reorganization are discussed.
Coexpression of lactosyl and ganglioteraosyl gangliosides in rat cerebellar radial glial cells in culture
Authors: Gravotta D. , Ferreira A. , Busciglio J. , Caceres A. , Landa C. A. , Maccioni H. J. F.
Published: January 2005

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotertrasylceramide (GTC) series on the surface of cells from rat embryonic cerebellar tissue was investigated by double‐color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosy‐GTC) was detected using the cholera toxin‐cholera toxin antibody experimental approach after conversion of polysialosyl‐GTC to GM1 by treatment of the cells with neuraminidase.

To distinguish the major neural cell types present in the cultures the expression of the following cell type‐specific markers was investigated: neurons‐specific markers was investigated: neuron‐specific enolase and microtubule‐associated protein‐2 (MAP‐2) as probes for neuronal cells and the intermediate filament protein glial fibrillar acidic protein (GFAP) as a probe for astroglial cells.

More than 80% of cells dissociated from cerebellar tissue of 15‐day‐old rat embryos (E15) are positive for the expression of GD3 and about 50% for the expression of GM1 and polysialosyl‐GTC, but most are negative for the expression of neuron‐specific enolase, MAP‐2, and GFAP. After culturing for 4 days (E15+4) most cells that show characteristics of neuronal cells are positive for the expression of polysialosyl‐GTC and “inactivate” the expression of GD3. Most cells with characteristics of radial and stellate glial cells are also positive for the expression of polysialosyl‐GTC, but unlike neuron‐like cells, they do not “inactivate” the expression of GD3.
Mechanical changes associated with synaptic transmission in the mammalian superior cervical ganglion
Authors: Kusano K. , Tasaki I.
Published: January 2005

Mechanical changes in the superior cervical ganglion (SCG) of the guinea pig, evoked by stimulating the pre‐or postganglionic nerve, were determined by using a mechano‐electric transducer. The mechanical change evoked by either one of these nerves was monophasic, an initial phase of rapid swelling (about 1 dyn/cm2) being followed by a slow relaxation. The mechanism of these mechanical changes was examined by treating the preparations with agents which are known to suppress or facilitate synaptic transmission in the SCG. Mechanical changes elicited by preganglionic nerve stimulation were suppressed by agents that reversibly block ganglionic transmission (Ca2+ ‐free high Mg2+, d‐tubocurarine, hexamethonium, TEA, TTX). On the other hand, agents which are known to facilitate synaptic transmission (high Ca2+) increased both the amplitude and the duration of swelling. In a Ba2+‐containing medium, a large, long‐lasting shrinkage of the ganglion was observed. It is suggested that the mechanical changes recorded in this study are associated with postsynaptic electrogenesis.
Heat generation associated with synaptic transmission in the mammalian superior cervical ganglion
Authors: Kusano K. , Tasaki I.
Published: January 2005

By use of a thermal detector constructed with a thin polyvinylidene fluoride film (PVDF), heat production in the superior cervical ganglion (SCG) of the guinea pig was examined. A single electric shock applied to the preganglionic nerve evokes a temperature rise of approximately 1.5 × 10−6 deg. The thermal responses summate when the preganglionic nerve is stimulated repetitively. The amplitude of the thermal response is increased when the preparation is treated with a high Ca2+ medium. Treatment with agents that block ganglionic transmission (high Mg2+, d‐tubocurarine, hexamethonium, TTX) reversibly suppresses thermal response. It is thus concluded that the thermal responses described in this paper are generated by the physio‐chemical events underlying postsynaptic electrogenesis in the SCG cells.
Spiny neurons lacking choline acetyltransferase immunoreactivity are major targets of cholinergic and catecholaminergic terminals in rat striatum
Authors: Pickel V. M. , Chan J.
Published: January 2005

The ultrastructural substrate for functional interactions between intrinsic cholinergic neurons and catecholaminergic afferents to the caudate‐putamen nucleus and nucleus accumbens septi (NAS) was investigated immunocytochemically. Single sections of glutaraldehyde‐fixed rat brain were processed (1) for the immunoperoxidase labeling of a rat monoclonal antibody against the acetylcholine‐synthesizing enzyme choline acetyltransferase (CAT) and (2) for the immunoautoradiographic localization localization of a rabbit polyclonal antiserum against the catecholamine‐synthesizing enzyme tyrosine hydroxylase (TH). The ultrastructural morphology and cellular associations did not significantly differ in the caudate‐putamen versus NAS. Immunoperoxidase reaction for CAT was seen in perikarya, dendrites, and terminals, whereas immunoautoradiography for TH was in terminals. The perikarya and dendrites immunolabeled for CAT were large, sparsely spiny, and postsynaptic mainly to unlabeled axon terminals. Only 2–3% of the CAT‐labeled terminals (n = 136) and less than 1% of the TH‐labeled terminals (n = 86) were apposed to, or formed synapses with, perikarya or dendrites immunoreactive for CAT. Most unlabeled and all labeled terminals formed symmetric synapses. In the same sample, 18% of the CAT and 16% of the TH‐labeled terminals were directly apposed to each other. Unlabeled dendritic shafts received the major (40% for CAT versus 23% for TH) synaptic input from cholinergic terminals, while unlabeled spines received the major (47% for TH versus 23% for CAT) synaptic input from catecholaminergic terminals. Neither the unlabeled dendrites or spines received detectable convergent input from CAT and TH‐labeled terminals. Thirteen percent of the CAT‐labeled and 14% of TH‐labeled terminals were in apposition to unlabeled terminals forming asymmetric, presumably excitatory, synapses with unlabeled dendritic spines. We conclude that in both the caudateputamen and NAS cholinergic and catecholaminergic terminals (1) form symmetric, most likely inhibitory, synapses primarily with non‐cholinergic neurons, (2) differentially synapse on shafts or spines of separate dendrites, and (3) have axonal appositions suggesting the possibility of presynaptic physiological interactions. These results support the hypothesis that the cholinergic‐dopaminergic balance in striatal function may be mediated through inhibition of separate sets of spiny projection neurons with opposing excitatory and inhibitory functions.
Low‐abundance 32‐kilodalton nuclear protein specifically enriched in the central nervous system
Authors: Shelton K. R. , Cunningham J. G. , Klann E. , Merchant R. E. , Egle P. M. , Bigbee J. W.
Published: January 2005

Recently, a low‐abundance nuclear protein, p32/6.3, has been identified in brain tissue (Egle and Shelton: Journal of Biological Chemistry 261:2294–2298,1986). Using a Western blot procedure, we describe its distribution in the nervous system, determine its relative enrichment in brain versus liver, kidney, and certain other tissues, and describe an isolation procedure from brain. Selective enrichment occurs in basal ganglia, diencephalon, hippocampus, cerebellum, brainstem, spinal cord, and cerebral cortex but not in retina, dorsal root ganglia, and sympathetic ganglia. Thus, enrichment is limited to areas of the central nervous system. p32/6.3 appears to be preferentially enriched in neurons, because in bulk‐isolated fractions from rat grey matter it is more abundant in neuron‐enriched fractions than in astrocyte‐enriched fractions. p32/6.3 is approximately 20‐fold more concentrated in an insoluble nuclear protein or matrix fraction from forebrain than from kidney, liver, adrenal gland, or retina. This degree of enrichment is an ancient trait, detectable in the chicken as well as mammals.
Cholinergic function in cultures of mouse spinal cord neurons
Authors: Wang F. Z. , Nelson P. G. , Fitzgerald S. C. , Hersh L. B. , Neale E. A.
Published: January 2005

Cholinergic synapeses formed in cultures of fetal mouse spinal cord (SC) and superior cervical ganglion (SCG) were studied using intracellular and extracellular stimulation and recording as well as immunohistochemical staining for choline acetylransferase (ChAT).

Dissociated SC neurons and SC explants exhibited cholinergic terminals on SCG and SC neurons as demonstrated by ChAT immunoreactivity. Intracellular recording s showed that cholinergic inputs to SCG neurons were relatively common and that these synaptic inputs were blocked by the nicotinic acetylcholine (ACh) receptor blocker, tubocurarine. A comparison of three preparatios indicated that the incidence of cholinergic activity recorded in SCG neurons was significantly higher in co‐cultures of SCG with spinal cord ventral horn (VH) neurons grown on a substrate of non‐neuronal cells from cerebral cortex, than in co‐cultures with VH alone or with SC and dorsla root ganglion cells. Consistency between cholinergic physiology and staining for ChAT‐positive terminals on SCG neuronal somata was obtained in cultures of SC explants grown with dissociated SCG.

Application of acetylcholine, muscarine, and /or vasoactive intestinal polypeptide (VIP) produced slow excitation of SC neurons. Fast excitatory cholinergic interactions between SC neurons were not observed. Excitatory synaptic interactions between SC neurons were augmented by ACh or muscarine, while inhibitory synaptic interactions were diminished. Both types of synaptic modulation probably were produced by a presynaptic mechanism. Acetylcholine or muscarine affected synbaptic interactions between SC neurons in only one‐third of the synaptic connections tested, suggesting that the incidence of presynaptically cholinoceptive SC neurons is low in dissociated cell cultures.

The experimental results show that a culture system incorporating dissociated fetal mouse SC neurons or explants of SC with sympathetic ganglion neurons expresses both nicotinic and muscarinic cholinergic function.
Different pathogenicity of encephalitic togaviruses in organotypic cultures of spinal cord slices
Authors: Shahar A. , Lustig S. , Akov Y. , David Y. , Schneider P. , Levin R.
Published: January 2005

The pathogenicity of two encephalitic Togaviruses, Sindbis virus (SV), an alphavirus, and West Nile virus (WNV), a flavivirus, was studied in organotypic cultures of fetal mouse spinal cord slices grown in roller tubes. After about 3 weeks in vitro, during which time the cultures became abundantly myelinated, they were infected either by 5 × 105 PFU SV or by 5 × 106 PFU WNV per culture. The viruses caused different patterns of cytopathogenicity: SV induced severe cytotoxicity in all glia cells and neurons with concomitant demyelination within 48 hr. In contrast, WNV, even 4 days after infection, caused only mild cytopathic effects mainly to neurons and astrocytes and a slight degree of damage to the myelin sheath. A most remarkable finding was the entrapment of WNV particles in the interperiod lines of the myelin sheaths. Treatment of cultures with mouse alpha and beta interferon prior to their infection with either virus protected the cultures from any viral damage. Long‐term exposure of non‐infected control organotypic cultures of fetal spinal cord slices to mouse interferons had no significant effect on neuronal and glial differentiation, and myelin formation.
Alzheimer disease proteins (A68) share epitopes with tau but show distinct biochemical properties
Authors: Ksiezak‐Reding H. , Binder L. I. , Yen Shu‐Hui
Published: January 2005

Alz 50, a monoclonal antibody raised against Alzheimer brain homogenate, reacts with neurofibrillary tangles, microtubule‐associated proteins tau, and Alzheimer brain proteins of molecular weight 70–60 kDa (A68). To study the relationship between A68 and normal human tau we compared the biochemical properties of these proteins and tested the reactivity of A68 with eight antibodies (Alz 50, Tau 60, Tau‐2, Tau 14, Tau‐1, Ab 636.7, NP14, Tau 46) that bind to various regions of tau molecule. On Western blots, all tau‐reactive antibodies, except Tau‐1, recognized A68. Pretreatment with alkaline phosphatase was required for the Tau‐1 binding to A68. A68 consisted of three polypeptides of 68, 64, and 60 kDa, while tau contained 4–6 polypeptides of 50–65 kDa. A68 was less heterogenous than tau in the number of pl variants on two‐dimensional gels. All A68 variants were more acidic (pl 5.5–6.5) than human tau (pl 6.5–8.5). Phosphatase treatment had only a minor effect on the pI and mobility of A68. Limited proteolysis of A68 with trypsin or chymotrypsin generated large fragments of 56–66 kDa (chymotrypsin) and 40–45 kDa (trypsin). While none of the fragments was recognized by Alz 50, the chymotryptic fragments were reactive with all the other tau antibodies, and the tryptic fragments were positive with five of the antibodies (Tau 14, Tau‐1, Ab 636.7, NP14, and Tau 46). The peptide maps of A68 differed from that of tau in the number and the size of the peptide fragments. The differences in biochemical properties of these proteins and the sharing multiple epitopes suggest that A68 is a modified form of tau. The modification in part may be due to phosphorylation, although other changes rendering different isoelectrical properties and susceptibility to proteases need to be considered. The removal of the Alz 50 epitope by a cleavage of a 2–3 kDa fragment which does not contain the most C‐terminal epitope (Tau 46) indicates that the Alz 50 epitope is located at the N‐terminal periphery of the A68 molecule.
Enzymatic activites during differentiation of the human neuroblastoma cells, LA‐N‐1 and LA‐N‐2
Authors: Singh I. N. , Sorrentino G. , McCartney D. G. , Massarelli R. , Kanfer J. N.
Published: January 2005

The presence of 10−5 M retinoec acid (RA) in the culture medium of LA‐N‐1, a catecholamonergic cell line, and LA‐N‐2, a cholinergic cell line, enhanced their morphological differentiation. Tyrosine hydroxylase (TH) activity of the LA‐N‐1 cell was increased in the RA‐treated cells compared with control cultures at day 4 and remained elevated. Choline actyltransferase (ChAT) activity in the LA‐N‐2 cells gradually increased until 8 days in vitro (DIV) voth in the untreated control and the RA treated cultures. This activity in control and treated cells decreased gradually to a constant level of activity. The ChAT activity at 8 DIV of RA‐treated La‐N‐2 cells was increased 2.1‐fold (P<0.001) as compared to the control cultures. This increase in ChAT activity was accompanied by a 73% decrease of acetylcholinesterase (AChE) activity in LA‐N‐2 cell by 8DIV. AChE activity of LA‐N‐1 cells was unchanged during the time course of the experiment. Phospholipase‐A2 (PL‐A2)activity in RA‐treated LA‐N‐2cells was increased at day 4 as compared with the control cultures. There were no differnces obseved in phopholipase‐D (PL‐D), choline kinase and FPC‐phosphodiesterases activities in RA‐treated and ‐untreated LA‐N‐1 and LA‐N‐2 cells.
Pineal gland as a model to elucidate the primary mode of action of alpha‐methyldopa: Alpha‐methyldopa induces an increase in the synthesis of N‐acetylserotoni and melatonin levels by the rat pineal gland
Authors: Welman A. D. , Daya S.
Published: January 2005

An attempt was made to use the pineal gland as a model for the study of the primary mode of action of alpha‐methyldopa, which is still unclear. Organ cultures of pineal glands from rats treated chronically with alpha‐methyldopa showed enhanced conversion or radio‐active serotonin to melatonin (aMT) as well as its precursor, N‐acetyl‐serotonin (aHT). This treatment was also found to raise serotonin‐N‐acetyltransferase (NAT) activity. Thse increases associated with alpha‐methyldopa treatment were further enhanced by the beta‐adrenergic agonist, isoproterenol, suggesting a supersensitivity‐type effect occurring at the level of the beta‐receptor. A subsequent binding study, however, showed a decrease in betareceptor binding with exposure to alpha‐methyldopa, providing mitigating eveidence against the occurrence of a supersensitivity phenomenon. It is possible that a metabolite of alpha‐methyldopa acts as an alpha 1 and beta‐agonist, resulting in greater melatonin (aMT) and N‐acetylserotonin (aHT) synthesis than by a beta‐agonist, isoproterenol.