bluuebunny/crossref_metadata_2025 huggingface dataset:
Monogalactosyl Diglyceride, a Marker for Myelination, Activates Oligodendroglial Protein Kinase C
Authors: Schmidt‐Schultz Tyede , Althaus Hans H.
Published: July 2010

Abstract: Protein kinase C (PKC) is activated by 1,2‐sn‐diacylglycerol (DAG), the source of which can either be phosphatidylinositol bisphosphate or phosphatidylcholine. Here, we show that monogalactosyl diglyceride (MGDG), a minor galactolipid present in oligodendrocytes (OLs) and myelin, which is designated as a marker for myelination, can enhance OL PKC activity. Based on different calcium and substrate requirements we conclude that MGDG and DAG activate different isoforms of PKC group A: MGDG primarily stimulates PKC‐α, and DAG primarily activates PKC‐γ. The presence of these PKC isoforms in OLs was confirmed by western blotting, whereas PKC‐β was only weakly stained, if at all. Addition of MGDG to the culture medium provided a higher density of regenerating OL fibers, which was not observed when membrane‐permeable DAG was used. These findings indicate that MGDG can modulate the OL PKC activity and that PKC‐α is the major PKC isoform involved in OL process formation.
Production and Characterization of a New Specific Antiserum Against the Taurine Putative Biosynthetic Enzyme Cysteine Sulfinate Decarboxylase
Authors: Almarghini K. , Barbagli B. , Tappaz M.
Published: July 2010

Abstract: We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion‐exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were im munostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.
Rapid Communication Isolation and Characterization of the Promoter of the Human GABAAReceptor α1 Subunit Gene
Authors: Kang Inwha , Lindquist David G. , Kinane T. Bernard , Ercolani Louis , Pritchard Gary A. , Miller Lawrence G.
Published: July 2010

Abstract:The GABAAreceptor, as assessed by ligand binding and chloride flux measurement in vivo and in vitro, is down‐regulated in response to chronic benzodiazepine exposure. The mRNA levels of the α1 and γ2 subunits of the receptor are also reduced. We have isolated the promoter of the gene encoding the α1 subunit of the GABAAreceptor to elucidate the regulatory mechanism of its expression. A DNA segment 650 bp long has been Isolated that includes 151 bp of untranslated 5’end of the cDNA sequence and 500 bp of potential promoter‐enhancer region. The transcriptional activity of this DNA segment linked to the firefly luciferase gene showed a strong orientation specificity. The promoter activity was localized to a 60‐bp segment by deletion mapping. Mobility shift binding assay results suggest that this segment may interact with one or more factors in HeLa cell nuclear extracts to form a transcriptional complex. Primary cultures of embryonic chick cortical cells transfected with the promoter‐luciferase construct were treated chronically with lorazepam. Transcriptional activity of this promoter construct was strongly repressed by chronic administration of lorazepam.
Action of Brefeldin A on Amphibian Neurons: Passage of Newly Synthesized Proteins Through the Golgi Complex Is Not Required for Continued Fast Organelle Transport in Axons
Authors: Smith Richard S. , Hammerschlag Richard , Snyder Richard E. , Chan Honey , Bobinski Judy
Published: July 2010

Abstract: The relation between the availability of newly synthesized protein and lipid and the axonal transport of optically detectable organelles was examined in peripheral nerve preparations of amphibia (Rana catesbeiana and Xenopus laevis) in which intracellular traffic from the endo‐plasmic reticulum to the Golgi complex was inhibited with brefeldin A (BFA). Accumulation of fast‐transported radio‐labeled protein or phospholipid proximal to a sciatic nerve ligature was monitored in vitro in preparations of dorsal root ganglia and sciatic nerve. Organelle transport was examined by computer‐enhanced video microscopy of single myelinated axons. BFA reduced the amount of radiolabeled protein and lipid entering the fast‐transport system of the axon without affecting either the synthesis or the transport rate of these molecules. The time course of the effect of BFA on axonal transport is consistent with an action at an early step in the intrasomal pathway, and with its action being related to the observed rapid (<1 h) disassembly of the Golgi complex. At a concentration of BFA that reduced fast‐transported protein by >95%, no effect was observed on the flux or velocity of anterograde or retrograde organelle transport in axons for at least 20 h. Bidirectional axonal transport of organelles was similarly unaffected following suppression of protein synthesis by >99%. The findings suggest that the anterograde flux of transport organelles is not critically dependent on a supply of newly synthesized membrane precursors. The possibilities are considered that anterograde organelles normally arise from membrane components supplied from a post‐Golgi storage pool, as well as from recycled retrograde organelles.
Activation of Nuclear Factor KB in Human Neuroblastoma Cell Lines
Authors: Körner Marie , Tarantino Nadine , Pleskoff Olivier , Lee Leo M. , Debré Patrice
Published: July 2010

Abstract: The nuclear factor KB (NF‐kB) is a eukaryotic transcription factor. In B cells and macrophages it is constitutively present in cell nuclei, whereas in many other cell types, NF‐KB translocates from cytosol to nucleus as a result of transduction by tumor necrosis factor α (TNFα), phorbol ester, and other polyclonal signals. Using neuro‐blastoma cell lines as models, we have shown that in neural cells NF‐KB was present in the cytosol and translocated into nuclei as a result of TNFa treatment. The TNFα‐activated NF‐KB was transcriptionally functional. NF‐KB activation by TNFα was not correlated with cell differentiation or proliferation. However, reagents such as nerve growth factor (NGF) and the phorbol ester phorbol 12‐myristate 13‐acetate (PMA), which induce phenotypical differentiation of the SH‐SY5Y neuroblastoma cell line, activated NF‐KB, but only in that particular cell line. In a NGF‐responsive rat pheochromocytoma cell line, PC12, PMA activated NF‐KB, whereas NGF did not. In other neuroblastoma cell lines, such as SK‐N‐Be(2), the lack of PMA induction of differentiation was correlated with the lack of NF‐kB activation. We found, moreover, that in SK‐N‐Be(2) cells protein kinase C (PKC) enzymatic activity was much lower compared with that in a control cell line and that the low PKC enzymatic activity was due to low PKC protein expression. NF‐KB was not activated by retinoic acid, which induced morphological differentiation of all the neuroblastoma cell lines used in the present study. Thus, NF‐KB activation was not required for neuroblastoma cell differentiation. Furthermore, the results obtained with TNFα proved that NF‐KB activation was not sufficient for induction of neuroblastoma differentiation.
In Vivo Evidence that Nonneuronal β‐Adrenoceptors as Well as Dopamine Receptors Contribute to Cyclic AMP Efflux in Rat Striatum
Authors: Suyama Kazuhiko , Dykstra Kevin H. , Masana Monica I. , Manji Husseini K. , Potter William Z.
Published: July 2010

Abstract:We applied in vivo microdialysis to assess the effects of dopaminergic and β‐adrenergic receptor stimulation on cyclic AMP efflux in rat striatum under chloral hydrate anesthesia. Dopamine (up to 1mM) infused for 20 min through the probe did not increase cyclic AMP, whereas both the selective dopamine D1agonist SKF 38393 and D2antagonist sulpiride produced modest increases. It is interesting that the β‐adrenoceptor agonist isoproterenol produced a marked increase (204.7% of basal level at 1 mM) which was antagonized by the β‐adreno‐ceptor antagonist propranolol. Pretreatment with a glial selective metabolic inhibitor, fluorocitrate (1mM), by a 5‐h infusion through the probe attenuated basal cyclic AMP efflux by 30.3% and significantly blocked the response to isoproterenol. By contrast, striatal injection of a neuro‐toxin, kainic acid (2.5 μg), 2 days before the dialysis experiment did not affect basal cyclic AMP or the response to isoproterenol, but blocked the response to SKF 38393. These data demonstrate that β‐adrenoceptors as well as dopamine receptors contribute to cyclic AMP efflux in rat striatum in vivo. They also suggest that basal and β‐adre‐noceptor‐stimulated cyclic AMP efflux are substantially dependent on intact glial cells.
Calcium Dependency and Tetrodotoxin Sensitivity of Neostriatal Dopamine Release in 5‐Day‐Old and Adult Rats as Measured by In Vivo Microdialysis
Authors: Gazzara Russell A. , Andersen Susan L.
Published: July 2010

Abstract:The calcium dependency and tetrodotoxin sensitivity of extracellular dopamine levels were assessed by microdialysis in the neostriatum of 5‐day‐old rat pups and were compared with those obtained in adult rats. The removal of calcium from the dialysate reduced spontaneous levels of extracellular dopamine to 20% of normal in the 5‐day‐old pups and to 10% of normal in the adults. Calcium‐free dialysate also decreased potassium‐evoked dopamine release to ∼20% of baseline in both ages. Furthermore, the addition of tetrodotoxin to the dialysate decreased spontaneous levels of extracellular dopamine to 10% of baseline in both ages. The effects of calcium removal and the addition of tetrodotoxin on extracellular levels of the dopamine metabolite 3,4‐dihydroxyphenylacetic acid were less pronounced. The results of this study demonstrate that extracellular levels of dopamine sampled by microdialysis in rats as young as 5 days of age are both calcium dependent and tetrodotoxin sensitive; thus, they are derived from neuronal activity and not from injury caused by acute implantation of the probe. Other age‐related differences support the hypothesis that dopamine release and turnover is greater In immature rats and may represent a form of compensation for incomplete dopamine nerve terminal ingrowth.
Estrogen Regulation of Dopamine Release in the Nucleus Accumbens: Genomic‐and Nongenomic‐Mediated Effects
Authors: Thompson Tina L. , Moss Robert L.
Published: July 2010

Abstract:The ability of estrogen to modulate mesolimbic dopamine (DA) was examined using in vivo voltammetry. Estrogen priming (5 μg, 48 h) of ovariectomized (ovx) female rats resulted in a slight decrease in K+‐stimulated DA release measured in the nucleus accumbens: this decrease was accompanied by a significant increase in both DA reuptake and DA clearance times. Following estrogen priming nomifensine, a potent inhibitor of the DA uptake carrier, was still able to potentiate K+‐stimulated DA release and alter the time course of DA availability, but the response was attenuated compared with ovx controls. Direct infusion of 17β‐estradiol hemisuccinate (17β‐E, 20–50 pg) into the nucleus accumbens resulted in a biphasic potentiation of K+‐stimulated release. An initial increase in release was observed 2 min after 17β‐E infusion; this increase, although reduced by 15 min, was still significantly higher than control values. A subsequent potentiation was observed 60 min after the initial 17β‐E infusion; this response remained for at least an additional 60 min. Nomifensine did not significantly alter K+‐stimulated DA release following 17β‐E infusion, but was still able to potentiate the total time DA was available extracellularly. These data suggest that the mesolimbic A10 DA neurons that terminate in the nucleus accumbens can be modulated in vivo by estrogen and that this modulation may be mediated by both genomic (long term) and nongenomic (short term) mechanisms.
Sequestration of Muscarinic Cholinergic Receptors in Permeabilized Neuroblastoma Cells
Authors: Slowiejko Diana M. , Levey Allan I. , Fisher Stephen K.
Published: July 2010

Abstract:The feasibility of using a permeabilized preparation of human SH‐SY‐5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin‐O, or the α‐toxin fromStaphylococcus aureusto oxotremorine‐M (Oxo‐M) for 30 min resulted in a 25–30% reduction in the number of cell surface mAChRs, as monitored by the loss ofN[3H]methyl‐ scopolamine ([3H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 μMdigitonin, the Oxo‐M‐mediated reduction in [3H]NMS binding was time (t1/2∼ 5 min) and concentration (EC50∼ 10 μM) dependent and was agonist specific (Oxo M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [3H]quinuclidinyl benzilate, occurred following Oxo‐M treatment. The loss of [3H]NMS sites observed in the presence of Oxo‐M was unaffected by omission of either ATP or Ca2+, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5′‐O‐(2‐thiodiphosphate). mAChRs sequestered in response to Oxo‐M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 μM). The results indicate (a) that permeabilized SH‐SY‐5Y cells support an agonist‐induced sequestration of mAChRs, the magnitude of which is ∼ 65–70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [3H]NMS has only a limited access despite the removal of the plasma membrane barrier, and (c) that the production of phosphoinositide‐derived second messengers is not a prerequisite for mAChR sequestration.
Growth of Ectopic Dendrites on Cortical Pyramidal Neurons in Neuronal Storage Diseases Correlates with Abnormal Accumulation of GM2 Ganglioside
Authors: Siegel Donald A. , Walkley Steven U.
Published: July 2010

Abstract: Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with ganglioside and nonganglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other gangliosides assayed for, including GM1 ganglioside. These results suggest a specific role for GM2 ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.
Expression of Polyubiquitin and Heat‐Shock Protein 70 Genes Increases in the Later Stages of Disease Progression in Scrapie‐Infected Mouse Brain
Authors: Kenward Nigel , Hope James , Landon Michael , Mayer R. John
Published: July 2010

Abstract: We have shown by northern analyses that the expression of the mouse polyubiquitin C gene is increased several fold in the brains of mice infected with both the ME7 and 87V strains of scrape. Expression of the polyubiquitin gene does not change significantly, compared with controls, until the later stages of disease progression when there is a 2.5‐fold increase in ME7‐infected brains and a 1.8‐fold increase in 87V‐infected brains. The patterns of changes of expression of the polyubiquitin genes in brains infected with the two strains of scrapie resemble those of accumulation of ubiquitin‐conjugate‐positive structures in the brain that are detected immunohisto chemically. A similar increase in the expression of a heatshock protein 70 gene also occurs.
Fate of Jimpy‐Type Oligodendrocytes in Jimpy Heterozygote
Authors: Kagawa Tetsushi , Nakao Junji , Yamada Masanobu , Shimizu Keiji , Hayakawa. Toru , Mikoshiba Katsuhiko , Ikenaka Kazuhiro
Published: July 2010

Abstract: In the jimpy mutant mouse, as well as in many other animals with mutations in the myelin proteolipid protein (PLP) gene, Oligodendrocytes degenerate before their maturation. To analyze whether this degeneration is caused by the loss of function of PLP gene products related to oligodendrocyte maturation/survival acting extrinsically, expression of the PLP gene was investigated in the jimpy heterozygote, in which one‐half of the cells are jimpy type and the other half are wild type due to random Xchromosome inactivation. We first showed that jimpy PLP gene expression is normally regulated at the early stages of development in brains of jimpy hemizygotes and heterozygotes, at least to day 2 after birth. However, the great increase in the level of PLP gene transcripts observed in wild‐type mouse brain is suppressed in jimpy mouse brain. This increase was also suppressed in the jimpy heterozygote, and by 2 months after birth, very few jimpy‐type PLP gene transcripts were detected in heterozygotes. These results indicate that jimpy‐type Oligodendrocytes cannot survive or are still in the immature stage in the brain of jimpy heterozygotes. Thus, degeneration of jimpy Oligodendrocytes is not caused merely by the lack of trophic factors.
Identification of a Peripherin Dimer: Changes During Axonal Development and Regeneration of the Rat Sciatic Nerve
Authors: Chadan S. , Moya K. L. , Portier M.‐M. , Filliatreau G.
Published: July 2010

Abstract: Western blotting of rat dorsal root ganglion (DRG) and sciatic nerve under nonreducing conditions revealed that a peripherin‐specific antibody recognized a protein species of 116/130 kDa, pi 5.6, in addition to peripherin (56 kDa, pl 5.6). We showed that this 116/130 kDa protein is a disulfide dimer of peripherin, because it gave rise to a single protein band comigrating with peripherin under reducing conditions and yielded the same proteolytic pattern as peripherin upon N‐chlorosuccinimide digestion. In addition, the immunological characteristics of the resulting peptides were identical to those of peripherin. We investigated the changes in peripherin monomer and dimer protein levels during axonal development and regeneration. During postnatal development, quantitative analysis of western blots of DRG proteins showed a significant increase in peripherin monomer (+52%) and dimer (+33%) levels from the day of birth [postnatal day 0 (PO)] to P7. The monomer levels remained high until P14 and then decreased so that at P21 and later ages, the monomer levels were similar to those observed at birth. In contrast, the dimer levels decreased continuously after P7, and in the adult, its level represented only 30% of the level at birth. Changes in [35S]methionine incorporation into adult DRG proteins were studied during regeneration of axotomized sciatic axons. Quantitative analysis of proteins showed a strong increase in labeling of both peripherin monomer (+56%) and dimer (+88%) 7 days after the crush. These levels, which remained high until 28 days after the axotomy, had returned to normal 70 days post axotomy. Our results show that peripherin monomer and dimer greatly increase during DRG fiber development and regeneration, suggesting that the two forms are involved in the growth of axons.
Stereotaxic Administration of 1‐Methyl‐4‐Phenylpyridinium Ion (MPP+) Decreases Striatal Fructose 2,6‐Bisphosphate in Rats
Authors: Espino Ana , Tortosa Avelina , Bendahan Gladys , Bartrons Ramon , Calopa Matilde , Ferrer Isidre , Ambrosio Santiago
Published: July 2010

Abstract:The Stereotaxic administration of 1‐methyl‐4‐phenylpyridinium ion (MPP+) into the neostriatum of male rats caused a lesion that resulted in a large dose‐dependent loss of striatal fructose 2,6‐bisphosphate; initial values were restored 5 days after the treatment. This effect was not protected by systemic administration of MK‐801 or by nitroarginine. The content of hexose 6‐phosphates and ATP was also reduced by MPP+treatment, whereas lactate was increased. Biochemical and histological results suggested that MPP+caused a nonselective cell death, followed by a pronounced astroglial response, parallel to fructose 2, 6‐bisphosphate recovery. The Stereotaxic administration of rotenone showed a different time effect on fructose 2,6‐bisphosphate cerebral content, with a significantly faster recovery. These results indicate that cerebral fructose 2,6‐bisphosphate may be a sensitive metabolite related to brain damage caused by potent neurotoxins such as MPP+. On the other hand, they show that MPP+acts in the brain through a quick, strong cytotoxic mechanism, which probably involves mechanisms other than mitochondria! chain blockage
Activities of Five Different Sialyltransferases in Fish and Rat Brains
Authors: Freischütz Bettina , Saito Megumi , Rahmann Hinrich , Yu Robert K.
Published: July 2010

Abstract: To investigate the role of Sialyltransferases in the metabolism of brain gangliosides, we examined activities of five different Sialyltransferases (GM3‐, GD3‐, GT3‐, GD1a‐, and GT1a‐synthase) using total membrane preparations from cichlid fish and Sprague‐Dawley rat brains, and analyzed the relationship between the enzyme activities and the ganglloside compositions. The patterns of sialyltransferase activities in fish and rat brains differed from each other. In fish brain, the GM3‐synthase activity was lower than GD3‐synthase activity, whereas the opposite relationship was observed in rat brain. The GT3‐synthase reaction with fish brain membranes produced radiolabeled GM3, GD3, and a ganglioside that was identified as GT3 based on mobility on TLC using two different solvent systems. No GT3‐synthase activity was detected in rat brain. The GD1a‐and GT1a‐synthase activities in fish brain were higher than those in rat brain. Although GT1a was a single radiolabeled ganglioside in fish GT1a‐synthase reaction, this ganglioside could not be detected in rat brain. The ratios of GM3‐, GD3‐, GT3‐, GD1a‐, and GT1a‐synthase activities in fish and rat brain were 23:31:4:28:14 and 61:21:0:18:0, respectively. Ganglioside analysis showed that fish brain was enriched with c‐series gangliosides including GT3 and polysialo‐species, whereas a‐and b‐se‐ries gangliosides were major components in rat brain. These results suggest that the species‐specific expression of gangliosides in brain tissues may be regulated, at least in part, at the level of sialyltransferase activities.
Photoendocrine Transduction in Cultured Chick Pineal Cells: IV. What Do Vitamin A Depletion and Retinaldehyde Addition Do to the Effects of Light on the Melatonin Rhythm?
Authors: Zatz Martin
Published: July 2010

Abstract: Light has at least two distinguishable effects on the circadian rhythm of melatonin output displayed by dispersed chick pineal cells in static culture: acute suppression of melatonin output and entrainment (phase shifts) of the underlying pacemaker. Previous results indicated that these two effects of light are mediated by different mechanistic pathways. The pathways for the acute and phase‐shifting effects of light either branch from the same, single photopigment or differ from the outset, starting from separate photopigments. If a single rhodopsin‐like photopigment mediates both effects of light, then vitamin A depletion and retinoid addition should affect both responses in parallel, although not proportionately. We therefore compared the effects of vitamin A depletion and retinoid addition on the acute and phase‐shifting effects of light under several experimental conditions. When chick pineal cells were depleted of vitamin A, acute responses to light were markedly reduced. Addition of 11‐cis‐retinaldehyde specifically restored (and enhanced) the acute response. When allowed to free run in constant red light, depleted cells displayed a rhythm of melatonin output with the same period as that of control cells. In contrast to the acute effects, phase shifts in response to 2‐ or 4‐h light pulses did not differ between depleted and control cells. Addition of retinaldehyde to depleted cells did not, by itself, reduce melatonin output or induce phase shifts. Retinaldehyde did increase the acute response to 4‐h light pulses but not the ensuing phase shifts. Responses increased with duration of the light pulse: Both the acute effect and the phase shifts induced by 4‐h light pulses were considerably larger than those induced by 2‐h (or 1‐h) light pulses. Addition of retinaldehyde to depleted cells increased the acute effect of 2‐h (or 1‐h) light pulses to at least that seen with 4‐h light pulses but did not Increase the size of the ensuing phase shifts. These results strongly confirm previous dissociations of the mechanistic pathways mediating the acute and phase‐shifting effects of light on chick pineal cells. They also support a role for rhodopsin‐like photopigment in the acute, but not phase‐shifting, response. They favor, but do not prove, the conclusion that separate photopigments mediate the acute and entraining effects of light.
Oxidation Products Arising from the Action of Monoamine Oxidase B on 1‐Methyl‐4‐Benzyl‐1,2,3,6‐Tetrahydropyridine, a Nonneurotoxic Analogue of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine
Authors: Sablin Sergey O. , Krueger Matthew J. , Bachurin Sergey O. , Solyakov Lev S. , Efange Simon M. N. , Singer Thomas P.
Published: July 2010

Abstract:1‐Methyl‐4‐benzyl‐1,2,3,6‐tetrahydropyridine (MBzTP), an analogue of the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine, despite its rapid oxidation by monoamine oxidase B (MAOB), is not neurotoxic. The pyridinium expected to arise from the four‐electron oxidation of MBzTP inhibits mitochondrial respiration and the oxidation of NADH in inner membranes and is only moderately less inhibitory than 1‐methyl‐4‐phenylpyridinium. It is also a competitive inhibitor of dopamine uptake by the dopamine transporter and hence likely to be taken up into neurons, despite its relatively highK1value (K1= 21 μM). Incubation of MBzTP with purified MAO B yields first the dihydropyridinium form, then a mixture of the pyridinium form and another unidentified product, in proportions that depend on the concentrations of MAO B and oxygen. At low MAO B concentration and moderate oxygen concentration, nonenzymatic formation, of the unidentified product predominates. The lack of neurotoxicity of MBzTP appears to be due to the oxidative destruction of the dihydropyridine and consequent failure of accumulation of 1‐methyl‐4‐behzylpyridinium.
Estimating Extracellular Concentrations of Dopamine and 3,4‐Dihydroxyphenylacetic Acid in Nucleus Accumbens and Striatum Using Microdialysis: Relationships Between In Vitro and In Vivo Recoveries
Authors: Glick S. D. , Dong N. , Keller R. W. , Carlson J. N.
Published: July 2010

Abstract: It is common practice in microdialysis studies for probes to be “calibrated” in artificial CSF and in vitro recoveries determined for all substances to be measured in vivo. Dialysate concentrations of such substances are then “corrected” for in vitro recoveries to provide “estimates” of extracellular concentrations. At least for dopamine, in vitro and in vivo recoveries are significantly different and, therefore, an estimate of extracellular dopamine based on correction for in vitro recovery is likely to be erroneous. Generally, however, the relative relationships of such estimates among animals are of interest rather than the “true” extracellular values. Such relationships would be valid to the extent that estimated values are correlated with or predictive of true values. Using the “no net flux” procedure, the present study sought to determine, for both dopamine and its metabolite 3,4‐dihydroxy‐phenylacetic acid (DOPAC), whether in vitro and in vivo recoveries would correlate with each other as well as whether respective estimated and true (no net flux) values of these substances would correlate with each other. Probes (3 mm; BAS/CMed MF‐5393), previously calibrated, were lowered into both the nucleus accumbens and striatum of freely moving rats the day before sample collection was begun. In vitro and in vivo recoveries were not significantly correlated (r= 0.1–0.3), for either dopamine or DOPAC. For both dopamine and DOPAC, however, there were significant correlations (r= 0.7–0.8) between estimated and true values. Surprisingly, when using these commercial probes, absolute dialysate levels for both substances were even better correlated (r = 0.9–0.95) with true values. This suggests that, with these probes, a direct comparison of dialysate concentrations can be used to determine relative changes in basal extracellular levels of dopamine and DOPAC when it is not practical to do no net flux studies (e.g., because of the time required to characterize a drug effect). The use of in vitro calibrations adjusts the values closer to the true values but also adds noise to each value and therefore should be avoided.
Evidence for a Distinct D1Like Dopamine Receptor that Couples to Activation of Phosphoinositide Metabolism in Brain
Authors: Undie Ashiwel S. , Weinstock Joseph , Sarau Henry M. , Friedman Eitan
Published: July 2010

Abstract:Dopamine and the D1, receptor agonist SKF 38393 activate the phospholipase C‐rnediated hydrolysis of phosphoinositides in brain slices. This action is selectively inhibited by SCH‐23390, thus suggesting its mediation through the dopamine D1receptor. To determine if the dopamine receptor that mediates Phosphoinositide hydrolysis is the adenylyl, cyclase‐linked D1receptor or a different subtype of the dopamine D1receptor, 20 benzazepine compounds that were previously characterized as selective dopamine D1receptor agonists were tested for stimulation of Phosphoinositide hydrolysis in rat striatal slices and for activation of adenylyl cyclase in rat striatal membranes. The compounds displayed a range of potencies and efficacies in stimulating adenylyl cyclase or Phosphoinositide hydrolysis. Compounds such as SKF 81427 and SKF 38393 were as efficacious as dopamine in stimulating Phosphoinositide hydrolysis, whereas other compounds, including SKF 85174 and SKF 86284, although showing high efficacy in stimulating cyclic AMP, failed to stimulate inositol phosphate formation. There was no correlation between the potencies (r= 0.016;p< 0.95) or efficacies (r=−0.294;p< 0.24) of the tested compounds in stimulating cyclic AMP formation and phosphoinositide hydrolysis. These observations indicate that the D1‐like dopamine receptor that mediates phosphoinositide hydrolysis is pharmacologically distinct from the classic D1receptor that is coupled to stimulation of cyclic AMP formation.
Cloning, Functional Coexpression, and Pharmacological Characterisation of Human cDNAs Encoding NMDA Receptor NR1 and NR2A Subunits
Authors: Le Bourdellès B. , Wafford K. A. , Kemp J. A. , Marshall G. , Bain C. , Wilcox A. S. , Sikela J. M. , Whiting P. J.
Published: July 2010

Abstract:Using expression cloning, and more recently using polymerase chain reaction cloning approaches, a family of ratN‐methyl‐d‐aspartate (NMDA) receptor subunit cDNAs has been described (NR1, NR2A, NR2B, NR2C, and NR2D). Here we report cloning and sequencing of cDNAs encoding isoforms of the human NR1 subunit (NR1a, NR1d, and NR1e) that differ at their C‐terminal end as a result of alternative splicing and also of a cDNA encoding the human NR2A subunit. The deduced amino acid sequences of the human NR1 subunit isoforms differed from the published rat NR1 subunit sequences at only eight positions, all of which were N‐terminal to the alternatively spliced domains. The human NR2A subunit deduced amino acid sequence differed from the published rat NR2A subunit sequence at 81 of the 1,464 amino acids, with most of the substitutions being located in the C‐terminal half of the subunit. The gene for NR2A has been localised to human chromosome 16. We also report the expression and pharmacological characterisation of recombinant human NR1a/NR2A heteromeric receptors inXenopusoocytes. These receptors had EC50values of 2.14 and 2.05 μMfor glutamate and glycine, respectively, and an IC50of 46.8 μMfor Mg2+. Responses were antagonised byd‐2‐amino‐5‐phosphonovalerate, L‐689,560, pH 6.3, zinc, and MK‐801. No modulatory effect was observed on application of ifenprodil, confirming previous observations with rat NR1 + NR2A recombinant receptors.
12‐O‐Tetradecanoylphorbol 13‐Acetate and Fibroblast Growth Factor Increase the 30‐kDa Substrate Binding Subunit of Type II Deiodinase in Astrocytes
Authors: Lennon Ana Maria , Esfandiari All , Gavaret Jean‐Michel , Courtin Françoise , Pierre Michel
Published: July 2010

Abstract: Type II 5′‐deiodinase (D‐II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3′‐triiodothyronine (T3) in the brain., The D‐II activity in astroglial cell cultures is induced by several pathways including cyclic AMP (cAMP), 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), and fibroblast growth factors (FGFs). We have examined the effect of TPA and FGFs on the 30‐kDa substrate binding subunit of D‐II, by affinity labeling with N‐bromoacetyl‐[128I]T4 in astroglial cells. TPA (0.1 μM), 20 ng/ml acidic FGF (aFGF), and 1 mM 8‐bromo cyclic AMP all caused an increase in the 30‐kDa protein. cAMP induced the greatest increase (fivefold) followed by TPA (3.2‐fold) and FGF (2.8‐fold). Glucocorticoids acted synergistically with cAMP and aFGF and promoted the effect of TPA. Affinity labeling was competitively inhibited by bromoacetyl‐T4 > bromoacetyl‐T3 > T4 > reverse T3 > iopanoic acid > T3 > 3,5,3‐triiodothyroacetic acid. The effect of TPA (0.1 μM) was maximum at 8 h and then gradually decreased. aFGF (20 ng/ml) plus heparin (17 μg/ml) induced a maximal 30‐kDa increase at 8 h, which stayed stable for up to 24 h. The effect of aFGF was concentration dependent. Of the other growth factors studied, only basic FGF and platelet‐derived growth factor induced small increases in the 30‐kDa protein. Epidermal growth factor had little effect. In vitro labeling of cAMP, TPA, and aFGF‐stimulated cell sonicates resulted in an increase in the 30‐kDa protein that paralleled the increase in D‐II activity. These results correlate well with our previous studies showing that several distinct signaling pathways regulate D‐II activity. They suggest that the regulation of D‐II in astrocytes by cAMP, TPA, and aFGF involves an accumulation of the 30‐kDa substrate binding subunit.
The Ability of Diphenylpiperazines to Prevent Neuronal Death in Dorsal Root Ganglion Neurons In Vitro After Nerve Growth Factor Deprivation and In Vivo After Axotomy
Authors: Eichler Marc E. , Dubinsky Janet M. , Tong Jianxin , Rich Keith M.
Published: July 2010

Abstract:The mechanism of neuroprotection by the calcium channel antagonist flunarizine against neuronal death is unknown. We investigated the ability of other calcium channel antagonists (cinnarizine, nimodipine, nicardipine, diltiazem, and verapamil), calmodulin antagonists, and calpain inhibitors to prevent neuronal death in rat dorsal root ganglion neurons in vitro after nerve growth factor (NGF) deprivation and the ability of cinnarizine and diltiazem to protect in vivo after axotomy. In vitro, only neurons treated with cinnarizine or flunarizine were protected from death after withdrawal. In vivo, cinnarizine, but not diltiazem, protected dorsal root ganglion neurons in rats after unilateral sciatic nerve crush. Intracellular calcium concentration ([Ca2+],) was evaluated with fura 2 after NGF deprivation In vitro. Neurons “committed to die” 24 h after NGF deprivation displayed a decline in [Caa+], before visible morphological deterioration consistent with cell death. The influx of extracellular calcium was not necessary to produce neuronal death. Neurons deprived of NGF gradually lost the ability to respond to elevated external potassium with an increase in [Ca2+], during the first 24 h after trophic factor deprivation. After 24 h, neurons deprived of NGF could not be rescued by readministration of NGF. Neurons protected from cell death with diphenylpiperazines maintained their response to high external potassium, suggesting continued membrane integrity. We speculate that diphenylpiperazines may protect sensory neurons via an unknown mechanism that stabilizes cell membranes.
Reactive Oxygen Species Influence Nerve Growth Factor Synthesis in Primary Rat Astrocytes
Authors: Naveilhan Philippe , Neveu Isabelle , Jehan Frédéric , Baudet Christel , Wion Didier , Brachet Philippe
Published: July 2010

Abstract: Newborn rat brain astrocytes, cultured in a serum‐free medium, were exposed for 30 min to two types of reactive oxygen species. Cells were either treated with the xanthine/xanthine oxidase (X/XOD) system, which generates both H2O2 and the O2βradical, or to H2O2 alone. Both treatments induced a dose‐dependent accumulation of nerve growth factor (NGF) transcripts, 6 h after the exposure. Maximal effect was obtained with 6 mU/ml XOD, or 10–4M H2O2. A rapid expression of protooncogenes of the jun and fos families was also noticed in X/XOD‐or H2O2‐treated cells. This phenomenon was transient in cells exposed to X/XOD. However, in the case of H2O2‐treated cells, the accumulation of c‐fos or c‐jun mRNAs was still pronounced 6 h after the end of the treatment and the levels of cell‐secreted NGF appeared relatively reduced, when compared with those obtained after a shock with the X/XOD system. This raised the possibility that H2O2 at 10–4M could depress protein synthesis. Measurements of the incorporation of radiolabeled amino acids into trichloroacetic acid‐precipitable material supported this assumption. Level of radioactivity associated with cellular material was dramatically reduced in H2O2‐treated cells, when it was compared with control or XI XOD‐trreated cells. Furthermore, treatment of cells with the protein synthesis inhibitor anisomycin had an effect similar to that of H2O2 because it caused an accumulation of c‐fos, c‐jun, and NGF transcripts after 6 h of treatment. It is concluded that the effect of H2O2 results from an impairment of protein synthesis, which is accompanied by the “superinduction” of c‐fos, c‐jun, or NGF genes. To understand better the effect of X/XOD, the reaction was conducted in the presence of a large excess of catalase, which removes H2O2. The presence of catalase reduced the amplitude of the response of the NGF gene to the X/XOD treatment. Conversely, low doses of H2O2 and X/ XOD were more efficient for the production of NGF than each treatment alone. These results suggest that both O2β and H2O2 are important metabolites in the process of activation of the NGF gene, possibly via the formation of the OH* radical or other reactive products, which could constitute active regulatory molecules.
Activation of Glutamate Receptors Stimulates the Formation of Nitrite in Synaptosomes from Rat Cerebellum
Authors: Gorbunov Nikolai , Esposito Ennio
Published: July 2010

Abstract:Synaptosomes from rat cerebellum were used to investigate the involvement of different glutamate receptor subtypes in the control of the synthesis of nitric oxide (NO), measured as its breakdown product nitrite (NO2‐). Synaptosomes incubated in the presence of NAD|PH andl‐arginine produced measurable levels of NO2‐, which were reduced by addition ofNω‐nitro‐l‐arginine methyl ester, an inhibitor of nitric oxide synthase. The selective ionotropic glutamate receptor agonistN‐methyl‐d‐aspartate (NMDA) induced a pronounced increase in NO2‐formation, which was prevented byNω‐nitro‐l‐arginine methyl ester and by the specific NMDA receptor antagonist Dl‐2‐amino‐5‐phosphonovaleric acid (AP‐5). The NMDA‐induced increase in NO2‐formation was blocked by chelation of extracellular Ca2+with EGTA. Bothl‐glutamate and the selective agonist for the metabotropic glutamate receptors (β)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid raised NO2‐production, which retumed to control levels after addition of Nω‐nitro‐l‐arginine methyl ester. The selective glutamate ionotropic receptor agonist (R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid did not cause any change in NO2formation. The stimulatory effect ofl‐glutamate was blocked by the metabotropic glutamate receptor antagonist Dl‐2‐amino‐4‐phosphonobutyric acid but was unaffected by the selective NMDA receptor blocker AP‐5. Removal of extracellular Ca2+by EGTA did not affect the action ofl‐glutamate; whereas W‐7, an inhibitor of calmodulin, and dantrolene, a compound that blocks the mobilization of Ca2+from intracellular stores, abolished the effect ofl‐glutamate on NO2‐formation. It is suggested that stimulation of ionotropic NMDA receptors activates NO metabolism by causing an influx of Ca2+from the extracellular space, whereas activation of metabotropic receptors byl‐glutamate provokes a mobilization of Ca2+from intracellular stores, which stimulates nitric oxide synthase activity by forning Ca2+/calmodulin complexes.
Forskolin and 3‐Isobutyl‐l‐Methylxanthine Increase Basal and Sodium Nitroprusside‐Elevated Cyclic GMP Levels in Adult Guinea‐Pig Cerebellar Slices
Authors: Hernàndez Félix , Alexander Stephen P. H. , Kendall David A.
Published: July 2010

Abstract:In this study, the interaction between 3′,5′‐cyclic adenosine monophosphate (cAMP) and 3′,5′‐cyclic guanosine monophosphate (cGMP) in [3H]adenine‐or [3H]‐guanine‐prelabelled adult guinea‐pig cerebellar slices was investigated. Basal levels of [3H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1‐100 μM). However, forskolin elicited a concentration‐dependent, saturable potentiation of sodium nitroprusside (SNP)‐stimulated [3H]cGMP accumulation (forskolin EC50value of 0.98 β 0.23 μM; 10 μMforskolin produced a 1.8 β 0.3‐fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001‐10 mM). forskolin also elicited a large stimulation of [3H]‐cAMP in [3H]adenine‐prelabelled guinea‐pig cerebellar slices; however, 1,9‐dideoxyforskolin failed to elicit either a [3H]cAMP response or a potentiation of the SNP‐induced [3H]cGMP response at concentrations up to 100 μM.Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. AG‐Nitro‐l‐arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP‐induced [3H]cGMP levels. The protein kinase inhibitors 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31‐8220 (10 μM) had no effect on the [3H]cGMP response to either SNP or the combination of SNP plus forskolin.N6,2′‐Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [3H]cGMP levels induced by SNP. 3‐lso‐butyl‐1‐methylxanthine reproduced the effect of forskolin on SNP‐induced [3H]cGMP levels, but a less‐than‐additive effect was observed when the response to SNP was studied in the presence of forskolin and 3‐isobutyl‐1‐methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea‐pig cerebellar slices, and that cAMP may regulate cGMP‐mediated responses in this tissue.
Pharmacological Evidence for the Involvement of Calciuml Calmodulin in Serotonin 5‐HT3Receptor‐Mediated Cation Permeability in NG 108‐15 Cells
Authors: Riad M. , Hamon M. , Emerit M. B.
Published: July 2010

Abstract:In NG 108–15 clonal cells, extracellular application of micromolar concentrations of serotonin [5‐hydroxy‐tryptamine (5‐HT)] and substance P induces the opening of a cation permeability monitored by the influx of [14C]‐guanidinium. The serotoninergic component of this cation permeability Is linked to 5‐HT3receptor activation, whereas the substance P component probably involves an “N‐terminal‐dependent substance P receptor.” In this study, [14C]guanidinium influx triggered by 1 μM5‐HT plus 10 μMsubstance P was shown to be insensitive to tetrodotoxin, verapamil, diltiazem, nimodipine, and ω‐conotoxin, as expected from a process independent of voltage‐sensitive sodium and calcium channels. In contrast, [14C]guanidinium influx was inhibited by millimolar concentrations of extracellular calcium and by the chelation of intracellular calcium by bis‐O‐aminophenoxyethanetetraacetic acid. The inhibition by extracellular calcium apparently involved a competition between the divalent cation and [14C]guanidinium for the same channel. When NG 108–15 cells were exposed to X537A, an ionophore that specifically induces release of calcium from intracellular stores, [14C]guanidinium uptake was markedly increased even in the absence of 5‐HT and/or substance P. Conversely, [14C]guanidinium influx due to the latter substances could be reversibly and dose‐dependently blocked by various drugs that possess calmodulin‐antagonizing properties. These results strongly suggest that the cation permeability opened by 5‐HT and substance P in NG 108–15 cells involves a calcium/calmodulin‐dependent process. However, as the phosphodiesterase inhibitor isobutylmethylxanthine, the nitric oxide synthase inhibitor A/monomethylarginine, the protein kinase C inhibitor staurosporine, and the protein kinase C activator 12‐O‐tetradeca‐noylphorbol 13‐acetate did not alter [14C]guanidinium influx in NG 108–15 cells exposed to 5‐HT and substance P., this process probably does not involve the calcium‐dependent nitric oxide pathway and protein kinase C activation.
Spontaneous and Amphetamine‐Evoked Release of Cerebellar Noradrenaline After Sensorimotor Cortex Contusion: An In Vivo Microdialysis Study in the Awake Rat
Authors: Krobert Kurt A. , Sutton Richard L. , Feeney Dennis M.
Published: July 2010

Abstract:Microdialysis sampling combined with HPLC was used to assess spontaneous andd‐amphetamine (AMPH)‐evoked release of noradrenaline (NA) in the cerebellum 1 day after probe implantation and 1 day after contusion of the right sensorimotor cortex (SMCX) in rats. In normal controls the mean β SEM basal NA release was 10.08 β 0.97 pg in the left cerebellar hemisphere and 8.21 β 1.17 pg in the right hemisphere 22–24 h after probe implantation. The average β SEM NA release in a 3‐h period after administration of AMPH (2 mg/kg, i.p.) increased to 453 β 47.35 pg in the left and to 402 β 49.95 pg in the right cerebellar hemisphere. NA release (range of 413–951% increase over baseline) was maximal 20–40 min postdrug, returned to basal levels within 5 h, and remained unchanged for the 22–24‐h postdrug measurement period. Animals with a focal SMCX contusion had a marked depression of both spontaneous and AMPH‐evoked NA release. Mean β SEM basal NA release was 4.84 β 1.09 pg in the left and 4.95 β 0.43 pg in the right cerebellar hemisphere from 22 to 24 h postinjury, with NA levels increasing to 259 β 75.44 and 219 β 23.45 pg in the respective hemispheres over a 3‐h period after AMPH. The maximal AMPH‐induced increase in NA release ranged from 522 to 1,088% of basal levels in contused rats, with NA release returning to predrug levels within 5 h and remaining depressed for at least 48 h postinjury. These data indicate that although neocortical injury results in a bilateral reduction of extracellular levels of NA in cerebellum, AMPH‐releasable NA stores are present in the cerebellum. These effects may be related to locomotor impairments and AMPH‐facilitated behavioral” recovery after cortical injury.