bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurochemical Characterization of Extracellular Serotonin in the Rostral Ventromedial Medulla and Its Modulation by Noxious Stimuli
Authors: Taylor Bradley K. , Basbaum Allan I.
Published: July 2010

Abstract: Using in vivo microdialysis, we have characterized serotonin release from the rostral ventromedial medulla of the freely moving rat. Addition of tetrodotoxin or removal of calcium from the dialysis solution diminished the dialysate serotonin content, suggesting that spontaneous, calcium channel‐ and sodium channel‐dependent neuronal release mechanisms contribute to the extracellular serotonin collected from the rostral ventromedial medulla. Extracellular serotonin concentration was increased by depolarization (with 100 mM potassium) and by the local administration of either a reuptake blocker (citalopram), a monoamine oxidase inhibitor (pargyline), or amphetamine. Serotonin release was reduced significantly by 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, suggesting that serotonin1A receptors may regulate release from rostral ventromedial medulla neurons. Because the basal serotonin concentration in the rostral ventromedial medulla was approximately twofold higher than that collected from the rostral ventrolateral medulla, a region that contains serotonin terminals but many fewer cell bodies, the possibility of release of serotonin from rostral ventromedial medulla neurons is discussed. Finally, intraplantar formalin injection significantly increased serotonin release, suggesting that this neurotransmitter contributes to nociceptive modulation by regulating the outflow of the rostral ventromedial medulla neurons.
Stimulation of Arginine Transport and Nitric Oxide Production by Lipopolysaccharide Is Mediated by Different Signaling Pathways in Astrocytes
Authors: Schmidlin Andreas , Wiesinger Heinrich
Published: July 2010

Abstract: Transport of l‐arginine and generation of nitrite in microglia‐free astroglial cultures derived from neonatal mouse brain were stimulated by bacterial lipopolysaccharide (LPS) in a time‐ and dose‐dependent manner. LPS stimulated arginine transport between 1.3‐ and 2.5‐fold; half‐maximal stimulation was obtained with 0.3 µg/ml LPS. Acceleration of transport was detectable within 6 h of incubation with LPS. Cycloheximide or actinomycin D neutralized the effect of LPS. Stimulation of generation of nitrite was reduced when the cells were incubated simultaneously with LPS and either genistein or diethyldithiocarbamate, inhibitors of protein tyrosine kinase and nuclear transcription factor κ, respectively. However, stimulation of arginine transport was not reduced in the presence of these compounds. Dexamethasone inhibited stimulation of nitric oxide (NO) production but not of arginine transport. Protein kinase C inhibitor staurosporine had no effect on either process. The results suggest that LPS‐stimulated acceleration of arginine transport in astrocytes requires protein as well as RNA synthesis. Induction of synthesis of an astroglial cationic amino acid transport system appears to be mechanistically independent from stimulation of intracellular NO production.
Sustained Dopamine Release Induced by Secretoneurin in the Striatum of the Rat: A Microdialysis Study
Authors: Agneter E. , Sitte H. H. , Stöckl‐Hiesleitner S. , Fischer‐Colbrie R. , Winkler H. , Singer E. A.
Published: July 2010

Abstract: Secretoneurin (SN) is a neuropeptide derived from secretogranin II that is found in brain and endocrine tissues. The aim of the present study was to determine the influence of this novel peptide on dopamine (DA) release from rat striatum using the microdialysis technique. Rat SN (1–30 µmol/L added to the dialysis buffer) enhanced DA outflow of awake rats in a concentration‐dependent way without marked effects on the outflow of 3,4‐dihydroxyphenylacetic acid or homovanillic acid. The increase in extracellular DA content caused by the peptide was observed throughout the entire period of administration (up to 4 h). Human SN and its 15‐amino‐acid C‐terminal sequence also increased DA outflow, but the effects were smaller than those of rat SN. Two other peptides derived from secretogranin II were without effect on DA efflux. These results establish that SN has a pronounced effect on DA release under in vivo conditions.
Changes in the Levels of Dolichol and Dolichyl Phosphate in a Murine Model of Niemann‐Pick's Type C Disease
Authors: Schedin S. , Pentchev P. G. , Brunk U. , Dallner G.
Published: July 2010

Abstract: The distributions of mevalonate pathway lipids in various organs of a mouse strain used as a model for Niemann‐Pick's type C disease were analyzed. Extensive accumulation of cholesterol was observed in all tissues with the exception of the brain, where the content of this lipid was decreased. The changes in total dolichol contents of most organs varied from a 50% decrease in the lung to a twofold increase in kidney and heart. There was relative enrichment of longer‐chain dolichols, but no increase in the relative amount of α‐unsaturated polyprenols was observed. The levels of dolichyl phosphate in all organs were increased, and most of this lipid was associated with bound oligosaccharides or proteins. Ubiquinone levels were largely unchanged. Subfractionation studies revealed that heavy and light lysosomes exhibited a 10‐fold increase in cholesterol level, the amount of dolichol was decreased in lysosomes and increased in microsomes, and there was an increase in the dolichyl phosphate levels of all three of these subfractions. These results indicate that in diseased mice cholesterol accumulation in various organs is paralleled by an increase in the dolichyl phosphate concentration, whereas dolichol transport from the endoplasmic reticulum to lysosomes is inhibited.
Clonidine Suppresses 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine‐Induced Reductions of Striatal Dopamine and Tyrosine Hydroxylase Activity in Mice
Authors: Fornai Francesco , Alessandrì Maria Grazia , Fascetti Flavia , Vaglini Francesca , Corsini Giovanni U.
Published: July 2010

Abstract: Recent findings have shown that excitatory amino acid may be involved in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) toxicity. At the same time, evidence is accumulating that the endogenous nor‐adrenergic system plays a protective role in MPTP‐induced striatal dopamine (DA) depletion and nigral dopaminergic cell death. Recently, α2‐adrenoceptors located on glutamatergic axons have been shown to inhibit glutamate overflow. In this study, we evaluated the effects of an α2‐agonist (clonidine) and an α2‐antagonist (yohimbine) on MPTP‐induced striatal DA depletion and tyrosine hydroxylase activity reduction. We show that clonidine is able to prevent the neurotoxicity of MPTP in mice. To exert this effect, clonidine (0.5 mg/kg) must be administered at least twice (30 min before and 30 min after MPTP). Administration of another α2‐agonist (detomidine, 0.3 mg/kg) attenuated the neurotoxicity induced by MPTP. We provide evidence that the protective effect obtained with clonidine was not due to decreased striatal content of 1‐methyl‐4‐phenylpyridinium (MPP+). We also show that yohimbine, which is a classic α2‐adrenoceptor antagonist with low affinity for imidazoline receptors, produced by itself an enhancement of MPTP toxicity and was able to block the protective effect of clonidine. These data raise the possibility that α2‐adrenoceptor may modulate the susceptibility of the nigrostriatal dopaminergic pathway to neurotoxicity.
Transferrin and Iron in Normal, Alzheimer's Disease, and Parkinson's Disease Brain Regions
Authors: Loeffler D. A. , Connor J. R. , Juneau P. L. , Snyder B. S. , Kanaley L. , DeMaggio A. J. , Nguyen H. , Brickman C. M. , LeWitt P. A.
Published: July 2010

Abstract: Oxidant‐mediated damage is suspected to be involved in the pathogenesis of several neurodegenerative disorders. Iron promotes conversion of hydrogen peroxide to hydroxyl radical and, thus, may contribute to oxidant stress. We measured iron and its transport protein transferrin in caudate, putamen, globus pallidus, substantia nigra, and frontal cortex of subjects with Alzheimer's disease (n = 14) and Parkinson's disease (n = 14), and in younger adult (n = 8) and elderly (n = 8) normal controls. Although there were no differences between control groups with regard to concentrations of iron and transferrin, iron was significantly increased (p < 0.05) in Alzheimer's disease globus pallidus and frontal cortex and Parkinson's disease globus pallidus, and transferrin was significantly increased in Alzheimer's disease frontal cortex, compared with elderly controls. The transferrin/iron ratio, a measure of iron mobilization capacity, was decreased in globus pallidus and caudate in both disorders. Regional transferrin and iron concentrations were generally more highly correlated (Pearson's correlation coefficient) in elderly controls than in Alzheimer's and Parkinson's disease. The altered relationship between iron and transferrin provides further evidence that a disturbance in iron metabolism may be involved in both disorders.
Phosphatase Activity Toward Abnormally Phosphorylated τ: Decrease in Alzheimer Disease Brain
Authors: Gong Cheng‐Xin , Shaikh Sadia , Wang Jian‐Zhi , Zaidi Tanweer , Grundke‐Iqbal Inge , Iqbal Khalid
Published: July 2010

Abstract: Microtubule‐associated protein τ is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated τ can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase‐1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward τ‐1 sites (Ser199/Ser202) using the hyperphosphorylated τ isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase‐2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of τ. The Km values of dephosphorylation of the hyperphosphorylated τ by protein phosphatase‐2A and 2B are similar. The τ phosphatase activity is decreased by ∼30% in brain of Alzheimer disease patients compared with those of age‐matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of τ in Alzheimer disease.
Phorbol Ester Binding Sites in the Fish Retina: Correlation with Stimulation of Endogenous Phosphorylation and Protein Kinase C Activation
Authors: Janssen‐Bienhold Ulrike , Buschmann‐Gebhardt Beate , Weiler Reto
Published: July 2010

Abstract: The injection of phorbol esters into the eyes of dark‐adapted teleost fish can mimic light effects in the retina and induces corresponding synaptic plasticity of horizontal cells (HCs). It is therefore very likely that protein kinase C (PKC) mediates light‐induced synaptic plasticity. In the present study, we investigated the distribution of PKC, the phorbol ester receptor, in isolated HCs and in the whole retina by using tritiated phorbol 12,13‐dibutyrate ([3H]PDBu). The binding characteristics analyzed for HC homogenates and retinal homogenates revealed that [3H]PDBu binding is time dependent, specific, saturable, and reversible. Binding sites in HCs displayed a dissociation constant of 11.5 nM and a total number of 2.8 pmol/mg of protein. Autoradiography revealed that [3H]PDBu labeling is present in all retinal layers, including HCs, where it is associated with the somata. Furthermore, the treatment with PDBu strongly affected the endogenous phosphorylation of several membrane, cytosolic, and HC proteins and led to PKC activation as measured by H1 histone phosphorylation. In HCs, the treatment with PDBu in particular affected the amount of 32P incorporated into a group of phosphoproteins (68, 56/58, 47, 28, and 15 kDa) that were recently shown to be affected by light adaptation. These proteins might therefore be considered as important components of the observed morphological and physiological synaptic plasticity of HCs in the course of light adaptation.
Selective Increase in Dopamine Utilization in the Shell Subdivision of the Nucleus Accumbens by the Benzodiazepine Inverse Agonist FG 7142
Authors: Horger Brian A. , Elsworth John D. , Roth Robert H.
Published: July 2010

Abstract: We examined the effects of the benzodiazepine inverse agonist FG 7142 on dopamine metabolism in the core and shell subdivisions of the nucleus accumbens. FG 7142 (15 mg/kg i.p.) or vehicle was administered to adult male rats 30 min before they were killed. Selected brain regions, including samples from the whole nucleus accumbens as well as core and shell subdivisions, were collected and assayed for tissue concentrations of dopamine and its major metabolite, 3,4‐dihydroxyphenylacetic acid. Consistent with previous reports, FG 7142 administration increased dopamine utilization in the medial prefrontal cortex but not the whole nucleus accumbens. Examination of subdivisions revealed that FG 7142 produced increased dopamine utilization in the shell subdivision of the nucleus accumbens. No effect of FG 7142 on dopamine utilization in the core region of the nucleus accumbens was observed. These data are discussed in terms of in vivo microdialysis studies reporting increased dopamine release in the nucleus accumbens after FG 7142 administration.
High‐Density Lipoprotein Inhibits UDP‐N‐Acetylgalactosamine:GM3,N‐Acetylgalactosaminyltransferase and Differentiation of Cultured Cerebral Cells: Comparison with a Formerly Described Inhibitor of This Enzyme
Authors: Kivatinitz Silvia C. , Grabois Viviana R. , Quiroga Santiago
Published: July 2010

Abstract: We have previously described a thermostable inhibitor of the UDP‐N‐acetylgalactosamine:GM3,N‐acetylgalactosaminyltransferase (GM2 synthase) purified from chicken blood serum. Some properties of the GM2 synthase inhibitory preparation (IP) resemble those of high‐density lipoprotein (HDL), i.e., both have a MW of 200,000 in native conditions and are resistant to denaturation by heat. These and other facts prompted us to test the possibility that lipoproteins regulate ganglioside biosynthesis in the CNS. For this purpose, serum lipoprotein fractions were isolated from chicken serum by flotation and were assayed as inhibitors of GM2 synthase activity and of neuron differentiation in culture. HDL (in contrast to fractions containing very low‐density or low‐density lipoprotein) inhibited GM2 synthase with the same specific activity as IP and inhibited neuron cell differentiation in culture in a similar way. Furthermore, these two preparations also share several other characteristics; i.e., both have the same cholesterol content, the same floating behavior on KBr gradients, and the same polypeptide pattern as detected by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and staining with Coomassie Blue, or after western blot and revealing with an antibody prepared against IP, which is able to diminish the inhibitory effect of this preparation. The results described indicate identity between HDL and IP and suggest that HDL (particularly apolipoprotein A) could play an important role on ganglioside biosynthesis modulation during CNS development. The antineuritogenic effect of HDL described in this study could be of physiological relevance during CNS development and response to injury.
A Calcium Channel from the Presynaptic Nerve Terminal of the Narke japonica Electric Organ Contains a Non‐N‐Type α2δ Subunit
Authors: Tokumaru Hiroshi , Shojaku Suzuyo , Takehara Hiroaki , Hirashima Naohide , Abe Teruo , Saisu Hideo , Kirino Yutaka
Published: July 2010

Abstract: A monoclonal antibody (MCC‐1) that recognizes the α2δ subunit complex of L‐type calcium channels from rabbit skeletal muscle membranes partially inhibited the evoked release of acetylcholine from synaptosomes isolated from the electric organ of the marine electric ray, Narke japonica. Digitonin extracts of synaptosomal plasma membranes were subjected to immunoaffinity column chromatography on MCC‐1‐Sepharose. The purified fraction contained a 170‐kDa protein that reacts with MCC‐1 and dissociates into smaller polypeptides under reducing conditions. In addition, immunoblotting analysis revealed the existence of syntaxin in the purified fraction, suggesting that the calcium channel forms a complex with syntaxin. However, MCC‐1 did not immunoprecipitate an ω‐conotoxin GVIA‐binding protein. These findings indicate that the 170‐kDa protein may be the α2δ subunit of a calcium channel that is distinct from the ω‐conotoxin GVIA‐sensitive N‐type calcium channel and partially responsible for the calcium influx that triggers the evoked release of acetylcholine.
Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices
Authors: Sarri Elisabet , Picatoste Fernando , Claro Enrique
Published: July 2010

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin‐1, and trans‐(1S,3R)‐aminocyclopentyl‐1,3‐dicarboxylic acid (trans‐ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi‐prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D‐catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin‐1, and trans‐ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 µM) was inhibited by the H1 receptor antagonist mepyramine with a Ki constant of 0.7 nM and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin‐1‐stimulated phospholipase D (EC50 44 nM) was not blocked by BQ‐123, a specific antagonist of the ETA receptor. Endothelin‐3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin‐1, and EC50 values of 16 and 3 nM, respectively. These results show that histamine and endothelin‐1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.
Glutamine from Glial Cells Is Essential for the Maintenance of the Nerve Terminal Pool of Glutamate: Immunogold Evidence from Hippocampal Slice Cultures
Authors: Laake Jon Henrik , Slyngstad Tove Anita , Haug Finn‐Mogens Šmejda , Ottersen Ole Petter
Published: July 2010

Abstract: The immunogold labeling for glutamate and glutamine was studied at the electron microscopic level in hippocampal slice cultures following inhibition of l‐glutamine synthetase [l‐glutamate:ammonia ligase (ADP‐forming); EC 6.3.1.2]. In control cultures, glutamate‐like immunoreactivity was highest in terminals, intermediate in pyramidal cell bodies, and low in glial cells. Glutamine‐like immunoreactivity was high in glial cells, intermediate in pyramidal cell bodies, and low in terminals. After inhibition of glutamine synthetase with l‐methionine sulfoximine, glutamate‐like immunoreactivity was reduced by 52% in terminals and increased nearly fourfold in glia. Glutamine‐like immunoreactivity was reduced by 66% in glia following l‐methionine sulfoximine, but changed little in other compartments. In cultures that were treated with both l‐methionine sulfoximine and glutamine (1.0 mM), glutamate‐like immunoreactivity was maintained at control levels in terminals, whereas in glia glutamate‐like immunoreactivity was increased and glutamine‐like immunoreactivity was decreased to a similar extent as in cultures treated with l‐methionine sulfoximine alone. We conclude that (a) glutamate accumulates in glia when the flux through glutamine synthetase is blocked, emphasizing the importance of this pathway for the handling of glutamate; and (b) glutamine is necessary for the maintenance of a normal level of glutamate in terminals, and neither reuptake nor de novo synthesis through pathways other than the glutaminase reaction is sufficient.
Parallel Induction of Nitric Oxide and Tetrahydrobiopterin Synthesis by Cytokines in Rat Glial Cells
Authors: Sakai Naoki , Kaufman Seymour , Milstien Sheldon
Published: July 2010

Abstract: Activation of monocyte‐derived macrophages with cytokines leads to the induction of nitric oxide synthase. Much less is known about the effects of cytokines on microglia, resident brain macrophages, or on astrocytes. In this study, we compared the induction by lipopolysaccharide, interferon‐γ, and tumor necrosis factor‐α of nitric oxide production and synthesis of tetrahydrobiopterin, the required cofactor for nitric oxide synthase, in microglia and peritoneal macrophages. Activation of microglia induced parallel increases in nitric oxide and intracellular tetrahydrobiopterin levels, although induction of the latter appears to be somewhat more sensitive to diverse stimulators. As with macrophages, inducible nitric oxide production in microglia was blocked by inhibitors of tetrahydrobiopterin biosynthesis. Interleukin‐2, an important component of the neuroimmunomodulatory system, was only a weak activator of microglia by itself but potently synergized with interferon‐γ to stimulate production of both nitric oxide and tetrahydrobiopterin. Astrocytes were also activated by lipopolysaccharide and combinations of cytokines but showed a somewhat different pattern of responses than microglia. Biopterin synthesis was increased to higher levels in astrocytes than in microglia, but maximal induction of nitric oxide production required higher concentrations of cytokines than microglia and the response was much lower. These results suggest that tetrahydrobiopterin synthesis in glial cells is a potential target for therapeutic intervention in acute CNS infections whose pathology may be mediated by overproduction of nitric oxide.
Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines
Authors: Maggio Roberto , Barbier Pascaline , Toso Andrea , Barletta Davide , Corsini Giovanni U.
Published: July 2010

Abstract: We have characterized the internalization of muscarinic acetylcholine receptors induced by the nitric oxide (NO)‐generating compound sodium nitroprusside. When Chinese hamster ovary cells, stably transfected with the human m4 muscarinic receptor subtype, were incubated for 1 h in the presence of 700 µM sodium nitroprusside, the number of receptors measured in intact cells with the hydrophilic ligand N‐[3H]methylscopolamine was reduced by 30%. The effect was dose dependent, beginning with a concentration of sodium nitroprusside as low as 45 µM. Removal of sodium nitroprusside from the incubation medium did not result in a recovery of the binding sites. The phenomenon was temperature dependent and was blocked by the muscarinic antagonist atropine. No receptor diminution was detected when the number of binding sites was evaluated with the lipophilic antagonist [3H]quinuclidinyl benzilate. This indicates that sodium nitroprusside induces a redistribution of the muscarinic receptors between the plasma membrane and an internal compartment of the cell. Receptor loss was readily reversed by treatment with the sulfhydryl reducing agent diethyldithiocarbamate. Our data provide evidence that muscarinic receptors are internalized by sodium nitroprusside through the oxidation of sulfhydryl groups; they also suggest that NO could play a role in muscarinic receptor desensitization.
Differential Expression of GABAA/Benzodiazepine Receptor Subunit mRNAs and Ligand Binding Sites in Mouse Cerebellar Neurons Following In Vivo Ethanol Administration: An Autoradiographic Analysis
Authors: Wu Chieh‐Hsi , Frostholm Adrienne , De Blas Angel L. , Rotter Andrej
Published: July 2010

Abstract: The γ‐aminobutyric acidA (GABAA)/benzodiazepine (BZ) receptor is a pentamer composed of subunits belonging to several classes (α1–6, β1–4, γ1–4, δ, and ρ1 and ρ2). In situ hybridization, radioligand autoradiography, and immunocytochemistry were used to examine GABAA/BZ receptor α1, α6, β2, β3, and γ2 subunit expression in murine Purkinje, granule, and deep cerebellar neurons after in vivo ethanol exposure. Chronic ethanol treatment resulted in decreased α1 subunit mRNA expression in each cell type, whereas the expression of α6 and γ2 subunit mRNA levels increased; no changes were observed in the expression of β2 and β3 subunit mRNA. GABA and BZ agonist binding and antibody staining paralleled the changes in mRNA levels. Acute ethanol injection resulted in increased expression of α1 and β3 mRNAs, whereas levels of α6, β2, and γ2 mRNAs remained stable. Our results indicate that, in cerebellar neurons, the expression of specific GABAA/BZ receptor subunit mRNAs, polypeptides, and binding sites is independently regulated by in vivo administration of alcohol. The observed changes were not restricted to any one cerebellar cell type, because subunit expression in Purkinje, granule, and deep cerebellar cells was similarly affected.
In Vivo Electrochemical Monitoring of Serotonin in Spinal Dorsal Horn with Nafion‐Coated Multi‐Carbon Fiber Electrodes
Authors: Rivot J.‐P. , Cespuglio R. , Puig S. , Jouvet M. , Besson J.‐M.
Published: July 2010

Abstract: Biosensors sensitive for in vivo monitoring of serotonin (5‐HT) in the CNS by differential normal pulse voltammetry were constructed by coating treated multi‐carbon fiber electrodes (mCFEs) with Nafion (N‐mCFE). In vitro sensitivities of mCFE and N‐mCFE were compared in solutions ranging from 5 nM to 20 µM of uric acid (UA), 5‐hydroxyindoleacetic acid (5‐HIAA), and 5‐HT. The mCFEs were three to seven times less sensitive for 5‐HIAA or UA than for 5‐HT. Nafion treatment dramatically decreased sensitivity for 5‐HIAA and UA of N‐mCFEs (∼103 times), whereas it remained in the nanomolar range for 5‐HT. In vivo, in the dorsal horn of the lumbar spinal cord of anesthetized rats, the monoamine oxidase inhibitor clorgyline (10 mg/kg i.p.) produced a reduction (55 ± 3% at 180 min) of peak 3 of oxidation current (characteristic of 5‐hydroxyindoles) monitored with mCFEs, but with N‐mCFEs (in this latter case the peak was termed 3N) peak 3N increased to 135 ± 5% at 180 min. The 5‐HT release‐inducer p‐chloroamphetamine (PCA; 6 mg/kg i.p.) induced a slight (12 ± 3% at 150 min) decrease in peak 3 measured with mCFEs, whereas with N‐mCFEs PCA induced a rapid increase of peak 3N (137 ± 6% at 90 min). The xanthine oxidase inhibitor allopurinol (10 mg/kg i.p.) produced a decrease (30 ± 3% at 180 min) in peak 3 (mCFEs), but peak 3N (N‐mCFEs) was not affected (106% at 180 min). After pretreatment with allopurinol, PCA also produced an increase (135 ± 6% at 90 min) in peak 3N. These in vitro and in vivo data provide evidence for a highly preferential detection of 5‐HT versus 5‐HIAA and UA by N‐mCFEs, which could be used to follow the extracellular 5‐HT concentration within very discrete structures throughout the CNS.
A Charybdotoxin‐Sensitive, Ca2+‐Activated K+ Channel with Inward Rectifying Properties in Brain Microvascular Endothelial Cells: Properties and Activation by Endothelins
Authors: Van Renterghem Catherine , Vigne Paul , Frelin Christian
Published: July 2010

Abstract: A charybdotoxin‐sensitive, Ca2+‐activated K+ channel was identified in cultured rat brain capillary endothelial cells by using conventional single‐channel recording techniques and 86Rb+‐influx and efflux experiments. Channel activity was dependent on the presence of Ca2+ on the cytosolic face of the membrane with a threshold concentration of 100 nM. It was inhibited by charybdotoxin (IC50 30 nM) and quinine (IC50 0.1 mM) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin‐1 (EC50 0.7 nM) and endothelin‐3 (EC50 7–10 nM). The actions of endothelins were prevented by BQ‐123 (Ki = 8 nM) in a competitive fashion, hence suggesting the involvement of an ETA‐receptor subtype. The channel activity was unaffected by cyclic AMP‐ or cyclic GMP‐elevating agents. The possible role of the intermediate conductance, Ca2+‐activated K+ channels for mediating K+ movements across the blood‐brain barrier is discussed.
Activation of Mitogen‐Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors
Authors: Kurino Masahito , Fukunaga Kohji , Ushio Yukitaka , Miyamoto Eishichi
Published: July 2010

Abstract: Mitogen‐activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non‐NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down‐regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca2+ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with 32P‐labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf‐1, the c‐raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC‐dependent and the Ca2+‐dependent pathways and that the activation of Raf‐1 is not involved.
Histamine‐Evoked Chromaffin Cell Scinderin Redistribution, F‐Actin Disassembly, and Secretion: In the Absence of Cortical F‐Actin Disassembly, an Increase in Intracellular Ca2+ Fails to Trigger Exocytosis
Authors: Zhang L. , Del Castillo A. Rodríguez , Trifaró J.‐M.
Published: July 2010

Abstract: Histamine is a known chromaffin cell secretagogue that induces Ca2+‐dependent release of catecholamines. However, conflicting evidence exists as to the source of Ca2+ utilized in histamine‐evoked secretion. Here we report that histamine‐H1 receptor activation induces redistribution of scinderin, a Ca2+‐dependent F‐actin severing protein, cortical F‐actin disassembly, and catecholamine release. Histamine evoked similar patterns of distribution of scinderin and filamentous actin. The rapid responses to histamine occurred in the absence of extracellular Ca2+ and were triggered by release of Ca2+ from intracellular stores. The trigger for the release of Ca2+ was inositol 1,4,5‐trisphosphate because U‐73122, a phospholipase C inhibitor, but not its inactive isomer (U‐73343), inhibited the increases in IP3 and intracellular Ca2+ levels, scinderin redistribution, cortical F‐actin disassembly, and catecholamine release in response to histamine. Thapsigargin, an agent known to mobilize intracellular Ca2+, blocked the rise in intracellular Ca2+ concentration, scinderin redistribution, F‐actin disassembly, and catecholamine secretion in response to histamine. Calphostin C and chelerythrine, two inhibitors of protein kinase C, blocked all responses to histamine with the exception of the release of Ca2+ from intracellular stores. This suggests that protein kinase C is involved in histamine‐induced responses. The results also show that in the absence of F‐actin disassembly, rises in intracellular Ca2+ concentration are not by themselves capable of triggering catecholamine release.
Regulation of DOPA Decarboxylase Activity in Brain of Living Rat
Authors: Cumming Paul , Kuwabara Hiroto , Ase Ariel , Gjedde Albert
Published: July 2010

Abstract: To test the hypothesis that l‐DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [3H]DOPA. The unidirectional blood‐brain clearance of [3H]DOPA (KD1) was 0.049 ml g−1 min−1. The relative DDC activity (kD3) was 0.26 min−1 in striatum, 0.04 min−1 in hypothalamus, and 0.02 min−1 in hippocampus. In striatum, 3,4‐[3H]dihydroxyphenylacetic acid ([3H]DOPAC) was formed from [3H]DA with a rate constant of 0.013 min−1, [3H]homovanillic acid ([3H]HVA) was formed from [3H]DOPAC at a rate constant of 0.020 min−1, and [3H]HVA was eliminated from brain at a rate constant of 0.037 min−1. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, kD3 was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.
Light‐Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells
Authors: Yoshida Kazuhiko , Imaki Junko , Matsuda Hidehiko , Hagiwara Masatoshi
Published: July 2010

Abstract: The signal pathway for light‐induced expression of c‐fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c‐fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c‐fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE‐binding protein (CREB) phosphorylation in retinal cells was examined with a phospho‐CREB‐specific antibody. Both flashing light and administration of the L‐type Ca2+ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c‐fos/SS mRNAs were expressed. These cells could be double‐stained with anti‐calmodulin kinase II (anti‐CaM kinase II) monoclonal antibody and phospho‐CREB‐specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser133 and CaM kinase II in the neural retina.
Metallothionein Induction in Neonatal Rat Primary Astrocyte Cultures Protects Against Methylmercury Cytotoxicity
Authors: Rising L. , Vitarella D. , Kimelberg H. K. , Aschner M.
Published: July 2010

Abstract: Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT‐I and MT‐II mRNAs were probed on northern blots with an [α‐32P]dCTP‐labeled synthetic cDNA probe specific for rat MT mRNA. MT‐I and MT‐II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, ∼550 and 350 bp for MT‐I and MT‐II, respectively. Expression of MT‐I and MT‐II mRNA in astrocyte monolayers exposed to 2 × 10−6M MeHg for 6 h was increased over MT‐I and MT‐II mRNA levels in controls. Western blot analysis revealed a time‐dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time‐dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic d‐[3H]aspartate uptake. Preexposure of astrocytes to CdCl2, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on d‐[3H]aspartate uptake that occurs in MeHg‐treated astrocytes with constitutive MT levels. Associated with CdCl2 treatment was a time‐dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl2 pretreatment) attenuate MeHg‐induced toxicity. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.
Evidence for Presynaptic Adenosine A2a Receptors Associated with Norepinephrine Release and Their Desensitization in the Rat Nucleus Tractus Solitarius
Authors: Barraco Robin A. , Clough‐Helfman Carolyn , Goodwin Bradley P. , Anderson Gordon F.
Published: July 2010

Abstract: Rat medullary brain segments containing primarily nucleus tractus solitarius (NTS) were used for superfusion studies of evoked transmitter release and for isotherm receptor binding assays. Isotherm binding assays with [3H]CGS‐21680 on membranes prepared from NTS tissue blocks indicated a single high‐affinity binding site with a KD of 5.1 ± 1.4 nM and a Bmax of 20.6 ± 2.4 fmol/mg of protein. The binding density for [3H]CGS‐21680 on NTS membranes was 23 times less than comparable binding on membranes from striatal tissue. Electrically stimulated (1 min at 25 mA, 2 ms, 3 Hz) release of [3H]norepinephrine ([3H]NE) from 400‐µm‐thick NTS tissue slices resulted in an S2/S1 ratio of 0.96 ± 0.02. Superfusion of single tissue slices with 0.1–100 nM CGS‐21680, a selective adenosine A2a receptor agonist, for 5 min before the S2 stimulus produced a significant concentration‐dependent increase in the S2/S1 fractional release ratio that was maximal (31.3% increase) at 1.0 nM. However, superfusion of tissue slices with CGS‐21680 over the same concentration range for 20 min before the S2 stimulus did not alter the S2/S1 ratio significantly from control release ratios. The augmented release of [3H]NE mediated by 1.0 nM CGS‐21680 with a 5‐min tissue exposure was abolished by 1.0 and 10 nM CGS‐15943 as well as by 100 nM 8‐(3‐chlorostyryl)caffeine, both A2a receptor antagonists, but not by 1.0 nM 8‐cyclopentyl‐1,3‐dipropylxanthine, the A1 receptor antagonist. Taken together, these results suggest that CGS‐21680 augmented the evoked release of [3H]NE in the NTS via activation of presynaptic A2a receptors within the same concentration range as the binding affinity observed for [3H]CGS‐21680. It was also apparent that this population of presynaptic adenosine A2a receptors in the NTS desensitized within 20 min because the augmenting action of CGS‐21680 on evoked transmitter release was not evident at the longer interval.
Opioid‐Induced Increase in [Ca2+]i in ND8‐47 Neuroblastoma × Dorsal Root Ganglion Hybrid Cells Is Mediated Through G Protein‐Coupled δ‐Opioid Receptors and Desensitized by Chronic Exposure to Opioid
Authors: Tang Tianlai , Kiang Juliann G. , Cote Thomas , Cox Brian M.
Published: July 2010

Abstract: δ‐Receptor agonists induce a concentration‐dependent increase in intracellular calcium concentration ([Ca2+]i) in ND8‐47 cells by activating dihydropyridine‐sensitive Ca2+ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [d‐Ser2,Leu5]enkephalin‐Thr (DSLET)‐induced increase in [Ca2+]i. Cholera toxin (10 nM, 24 h) had no effect on DSLET‐induced response. Pretreatment of the cells with 1 µM DSLET for 1 h resulted in a 30% inhibition of DSLET‐induced increase in [Ca2+]i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 µM DSLET for 24 h demonstrate a nearly 90% decrease in [3H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits αi2, αi3, αs, and αq were detected in ND8‐47 cell membranes by western blot; αo and αi1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the α‐subunits. These results suggest that the DSLET‐induced increase in [Ca2+]i is mediated through pertussis toxin‐sensitive G proteins (probably Gi2 or Gi3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.
Phosphorylation and Agonist‐Specific Intracellular Trafficking of an Epitope‐Tagged μ‐Opioid Receptor Expressed in HEK 293 Cells
Authors: Arden James R. , Segredo Veronica , Wang Zaijie , Lameh Jelveh , Sadée Wolfgang
Published: July 2010

Abstract: We expressed the cloned μ‐opioid receptor (μR) in high abundance (5.5 × 106 sites/cell) with an amino‐terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope‐tagged receptor (EE‐μR) was similar to the untagged μR in ligand binding and agonist‐dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the μ‐specific peptide agonist [d‐Ala2,MePhe4,Glyol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist‐induced cellular redistribution of EE‐μR. Tracer binding studies suggested partial net internalization and a small degree of down‐regulation caused by DAMGO. EE‐μR‐containing membranes were solubilized in detergent [3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate] and immunoprecipitated by an anti‐epitope monoclonal antibody. Immunoblotting revealed a prominent band at ∼70 kDa with weaker bands at ∼65 kDa. EE‐μR was labeled with [γ‐32P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65–70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced ∼1.8‐fold with the addition of morphine. In conclusion, intracellular trafficking of the μR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein‐coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.
Acute Stimulatory Effect of Estradiol on Striatal Dopamine Synthesis
Authors: Pasqualini Catherine , Olivier Valérie , Guibert Bernard , Frain Odile , Leviel Vincent
Published: July 2010

Abstract: The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17α or 17β E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose‐dependent increase in striatal TH activity was observed within minutes after 17β (but not 17α) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push‐pull cannula supplied with an artificial CSF containing [3H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) were measured at 20‐min intervals. Addition of 10−9M 17β (but not 17α) E2 to the superfusing fluid immediately evoked an ∼50% increase in [3H]DA and [3H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2‐induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (Ki DA) was calculated. Incubation of striatal slices in the presence of 10−9M 17β (but not 17α) E2 indeed evoked an approximate twofold increase in the Ki DA of one form of the enzyme. It is concluded that physiological levels of E2 can act directly on striatal tissue to stimulate DA synthesis. This stimulation appears to be mediated, at least in part, by a decrease in TH susceptibility to end‐product inhibition, presumably due to phosphorylation of the enzyme. The rapid onset of this effect, and the fact that the striatum does not contain detectable nuclear E2 receptors, suggest a nongenomic action of the steroid.
τ Protein from Alzheimer's Disease Patients Is Glycated at Its Tubulin‐Binding Domain
Authors: Ledesma M. Dolores , Bonay Pedro , Avila Jesús
Published: July 2010

Abstract: Glycated residues of τ protein from paired helical filaments isolated from the brains of Alzheimer's disease patients were localized by doing a proteolytic cleavage of the protein, fractionation of the resulting peptides, and identification of those peptides using specific antibodies. The most suitable residues for glycation, lysines, present at the tubulin‐binding motif of τ protein, seem to be preferentially modified compared with those lysines present at other regions. Among these modified lysines, those located in the sequence comprising residues 318–336 (in the largest human τ isoform) were found to be glycated, as determined by the reaction with an antibody that recognizes a glycated peptide containing this sequence. Because those lysines are present in a tubulin binding motif of τ protein, its modification could result in a decrease in the interaction of τ with tubulin.
Convulsant Agents Activate c‐fos Induction in Both a Calmodulin‐Dependent and Calmodulin‐Independent Manner
Authors: Barrón S. , Tusell J. M. , Solà C. , Serratosa J.
Published: July 2010

Abstract: Calcium acts as a second messenger and can enter neurons through several types of calcium channel. We sought to determine whether the calcium‐dependent mechanisms inducing c‐fos expression are identical following activation, by appropriate drugs, of L‐type voltage‐sensitive calcium channels or NMDA and non‐NMDA receptors or following inhibition of the GABAergic system. We used primary cortical neurons and OF1 mice, and the levels of c‐fos protein and c‐fos mRNA were detected after treatment with the drugs by means of immunocytochemistry and in situ hybridization. The calmodulin antagonist N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7) abolished γ‐hexachlorocyclohexane‐, Bay K 8644‐, pentylenetetrazole‐, and kainic acid‐induced increases in c‐fos expression in cultured neurons. Conversely, W‐7 did not affect either NMDA‐ or picrotoxinin‐mediated increases in c‐fos expression. In mice, the pattern of protooncogene expression displayed some differences compared with cultured neurons, depending on the treatment. W‐7 administered before γ‐hexachlorocyclohexane, Bay K 8644, or pentylenetetrazole blocked the expression of c‐fos elicited by these compounds. However, W‐7 was not able to abolish c‐fos expression induced by picrotoxinin. In the animals treated with W‐7 before kainic acid or NMDA administration, c‐fos expression was inhibited in cerebral cortex, but it was still present in hippocampus. These results agree with the existence of diverse mechanisms transducing the calcium signals to the nucleus. Calmodulin may mediate neuronal responses depending on the route by which calcium enters the neuron, resulting in activation of different enzymes.