bluuebunny/crossref_metadata_2025 huggingface dataset:
Haloperidol and Fluphenazine Induce junB Gene Expression in Rat Striatum and Nucleus Accumbens
Authors: Simpson C. S. , Morris B. J.
Published: July 2010

Abstract: Administration of neuroleptics such as haloperidol to rats is known to induce the expression of the immediate‐early genes (IEGs) c‐fos and zif/268 in striatal neurones. Another IEG, junB, is of interest because it may be involved in the suppression, rather than the enhancement, of downstream gene transcription. In this study, rat striatal tissue was assayed for IEG expression by in situ hybridisation, after the injection of haloperidol (1 mg/kg) or fluphenazine (3 mg/kg). In addition to c‐fos mRNA and zif/268 mRNA, neurones in both the striatum and nucleus accumbens were found to contain high levels of junB mRNA, after treatment with either haloperidol or fluphenazine. The proportion of striatal neurones expressing junB mRNA strongly suggests that induction occurs in striatal projection neurones. A significant increase in the levels of the mRNA encoding another IEG, junD, was also detected after haloperidol treatment. The atypical neuroleptic clozapine (3 mg/kg) did not induce the expression of any of these IEGs in striatal tissue. The results show additional complexity in the pattern of IEG induction after neuroleptic administration and suggest that junB is involved in mediating some of the effects of neuroleptics on striatal gene expression.
The Metabotropic Glutamate Receptor mGluR4, but Not mGluR1α, Is Palmitoylated when Expressed in BHK Cells
Authors: Alaluf Simon , Mulvihill Eileen R. , McIlhinney Robert A. J.
Published: July 2010

Abstract: Several G protein‐coupled receptors have been shown to be palmitoylated, and for some of these receptors the covalent attachment of palmitate has been implicated in the regulation of receptor‐G protein coupling. The metabotropic glutamate receptor (mGluR) family forms a distinct group of G protein‐coupled receptors, and the possibility that these may also be palmitoylated has been examined. Clonal baby hamster kidney (BHK) cells permanently transfected with the mGluR4 and mGluR1α subtypes were labelled with [3H]palmitic acid. The cells were lysed, the receptors were immuno‐precipitated with specific antipeptide antibodies, and the immunoprecipitates were analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and autoradiography. The palmitoylated, endogenously expressed G protein α‐subunit αq could be immunoprecipitated from [3H]palmitate‐labelled BHK cells expressing mGluR1α using a specific antipeptide antibody, but in the same cell lysates no detectable [3H]palmitate‐labelled mGluR1α was found. This suggests that this mGluR subtype, associated with stimulation of phospholipase C, is not palmitoylated. In contrast, mGluR4, which is coupled to inhibition of adenylyl cyclase, was found to be labelled with [3H]palmitic acid, and the palmitate was quantitatively removed by treatment with 1 M hydroxylamine, suggesting attachment of the palmitate through a thioester bond. Stimulation with maximal doses of the neurotransmitter glutamate for 1, 5, or 10 min appeared to have no effect on the level of receptor palmitoylation.
Altered Cholinergic Metabolism in Rat CNS Following Aluminum Exposure: Implications on Learning Performance
Authors: Julka Deepinder , Sandhir Rajat , Gill Kiran Dip
Published: July 2010

Abstract: The effects of Al on the central cholinergic system have been studied. Al, at a dose of 10 mg/kg of body weight/day for 4 weeks, had a deleterious effect on the activities of biosynthetic (choline acetyltransferase) and hydrolytic (acetylcholinesterase) enzymes of the neurotransmitter acetylcholine. The levels of acetylcholine were also significantly lowered in different brain regions at the end of the dose regimen. There was a significant decrease in high‐affinity choline uptake following Al exposure. Muscarinic acetylcholine receptor binding studies revealed a decreased number of binding sites (Bmax), with the maximum effects being manifested in the hippocampus. Exogenous addition of 10 µM desferrioxamine restored the muscarinic receptor binding completely. The impaired cholinergic functioning had severe effects on cognitive functions. Neurobehavioral deficits were manifested in terms of decreased active (52%) and passive (73.30%) avoidance tests. The results suggest that Al exerts its toxic effects by altering cholinergic transmission, which is ultimately reflected in neurobehavioral deficits.
Modulation in Acetylcholinesterase of Rat Brain by Pyrethroids In Vivo and an In Vitro Kinetic Study
Authors: Rao Gazula Valeswara , Jagannatha Rao Kosagi Sharaf
Published: July 2010

Abstract: The modulation in acetylcholinesterase (AChE) of rat brain by two pyrethroids—permethrin (PM) and cypermethrin (CPM)—was studied both in vivo and in vitro. PM inhibited AChE activity in all regions of the rat brain (cerebral cortex, cerebellum, corpora striata, brainstem, hippocampus, and hypothalamus) at 4, 8, and 12 h after gastric intubation, whereas CPM elevated the enzyme activity in vivo. Substrate‐dependent enzyme kinetic studies have shown that PM and CPM behave as mixed‐type inhibitors, as evidenced by alterations in both Michaelis‐Menten constant (Km) and maximal velocity (Vmax) values. This indicates that both PM and CPM and substrate acetylcholine interact at hydrophobic subsites and may be able to bind simultaneously to the enzyme.
The UDP‐Galactose:Ceramide Galactosyltransferase: Expression Pattern in Oligodendrocytes and Schwann Cells During Myelination and Substrate Preference for Hydroxyceramide
Authors: Schaeren‐Wiemers N. , Van Der Bijl P. , Schwab M. E.
Published: July 2010

Abstract: Galactosylceramide (“galactocerebroside”; GalC) is a major glycolipid in the myelin sheath of the CNS and the PNS. The enzyme UDP‐galactose:ceramide galactosyltransferase (CGalT) catalyzes the final step of the synthesis of GalC: the transfer of galactose to ceramide. By a differential screening approach, we have isolated a cDNA, the sequence of which is identical to the recently isolated cDNA clones for CGalT. By northern analysis and in situ hybridization we demonstrated that CGalT mRNA is expressed at birth in oligodendrocytes and Schwann cells, an expression pattern corresponding to the onset of myelination. In addition to the high expression levels of CGalT in oligodendrocytes and Schwann cells, in situ hybridization also showed expression in subtypes of neurons in spinal cord, cerebellum, and brainstem in the adult CNS, but at a much lower level than in oligodendrocytes. Expression of CGalT in COS cells demonstrated that CGalT has a preference for hydroxyceramide as a substrate. CGalT‐expressing COS cells synthesize and transport GalC to their cell surface as shown by immunofluorescence and by lipid analysis of living cells. Our results suggested that the CGalT specifically uses hydroxyceramide for the synthesis of GalC and that separate (co)enzymes are not needed.
Effects of NO‐Generating Compounds on Synaptosomal Energy Metabolism
Authors: Erecińska Maria , Nelson David , Vanderkooi Jane M.
Published: July 2010

Abstract: The effects of nitroprusside and S‐nitrosocysteine, compounds that generate nitric oxide (NO), on synaptosomal energy‐producing pathways and energy level were investigated. The decrease in respiration was much faster and more pronounced with S‐nitrosocysteine than with nitroprusside. S‐Nitrosocysteine, at 10 µM, inhibited by 80% respiration with glucose and succinate (plus rotenone) in intact synaptosomes and with ascorbate/cytochrome c in broken preparations. Oxygenated hemoglobin reversed and/or prevented the inhibition, whereas glutathione (GSH) prolonged it. Under aerobic conditions, the synaptosomal energy level (creatine phosphate/creatine and ATP/ADP ratios) was reduced by the presence of S‐nitrosocysteine, whereas lactate generation was enhanced. The effects on energy parameters were greater at 5 min than at 15 min of incubation and were more pronounced in the presence of GSH. Under strictly anaerobic conditions, lactate production was reduced by the NO‐generating compounds in a concentration‐dependent manner. It is concluded that (a) inhibition of oxidative phosphorylation by NO leads to a fall in the synaptosomal energy level, which in turn stimulates glycolysis; (b) glycolysis can be inhibited by higher concentrations of the radical; and (c) inhibitory effects on the energy‐generating pathway and ATP level could contribute to NO toxicity under some in vivo situations.
Protein Phosphatase 2A Is the Major Enzyme in Brain that Dephosphorylates τ Protein Phosphorylated by Proline‐Directed Protein Kinases or Cyclic AMP‐Dependent Protein Kinase
Authors: Goedert M. , Jakes R. , Qi Z. , Wang J. H. , Cohen P.
Published: July 2010

Abstract: The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule‐associated protein τ. Previous studies have identified serine and threonine residues phosphorylated in PHF‐τ and have shown that τ can be phosphorylated at several of these sites by proline‐directed protein kinases and cyclic AMP‐dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant τ phosphorylated with mitogen‐activated protein kinase, glycogen synthase kinase‐3β, neuronal cdc2‐like kinase, or cyclic AMP‐dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates τ phosphorylated in this manner.
Muscarinic Receptor Sequestration in SH‐SY5Y Neuroblastoma Cells Is Inhibited when Clathrin Distribution Is Perturbed
Authors: Slowiejko Diana M. , McEwen Edward L. , Ernst Stephen A. , Fisher Stephen K.
Published: July 2010

Abstract: The possibility that clathrin plays a role in the agonist‐mediated sequestration of muscarinic cholinergic receptors in human SH‐SY5Y neuroblastoma cells has been investigated by the application of experimental paradigms previously established to perturb clathrin distribution and receptor cycling events. Preincubation of SH‐SY5Y cells under hypertonic conditions resulted in a pronounced inhibition of agonist‐induced muscarinic receptor sequestration (70–80% at 550 mOsm), which was reversed when cells were returned to isotonic medium. Depletion of intracellular K+ or acidification of the cytosol also resulted in >80% inhibition of muscarinic receptor sequestration. Under conditions of hypertonicity, depletion of intracellular K+, or acidification of cytosol, muscarinic receptor‐stimulated phosphoinositide hydrolysis and Ca2+ signaling events were either unaffected or markedly less inhibited than receptor sequestration. That these same experimental conditions did perturb clathrin distribution was verified by immunofluorescence studies. Hypertonicity and depletion of intracellular K+ resulted in a pronounced accumulation of clathrin in the perinuclear region, whereas acidification of the cytosol resulted in the appearance of microaggregates of clathrin throughout the cytoplasm and at the plasma membrane. The results are consistent with the possibility that muscarinic receptors in SH‐SY5Y cells are endocytosed via a clathrin‐dependent mechanism.
Serotonin 5‐HT1D and 5‐HT1A Receptors Respectively Mediate Inhibition of Glutamate Release and Inhibition of Cyclic GMP Production in Rat Cerebellum In Vitro
Authors: Maura Guido , Raiteri Maurizio
Published: July 2010

Abstract: The K+‐evoked overflow of endogenous glutamate from cerebellar synaptosomes was inhibited by serotonin [5‐hydroxytryptamine (5‐HT); pD2 = 8.95], 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT; pD2 = 7.35), and sumatriptan (pD2 = 8.43). These inhibitions were prevented by the selective 5‐HT1D receptor antagonist N‐[4‐methoxy‐3‐(4‐methyl‐1‐piperazinyl)phenyl]‐2′‐methyl‐4′‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)(1,1‐biphenyl)‐4‐carboxamide (GR‐127935). The three agonists tested also inhibited the cyclic GMP (cGMP) response provoked in slices by K+ depolarization; pD2 values were 9.37 (5‐HT), 9.00 (8‐OH‐DPAT), and 8.39 (sumatriptan). When cGMP formation was elevated by directly activating glutamate receptors with NMDA or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA), the inhibition of the cGMP responses displayed the following pattern: 5‐HT (pD2 values of 8.68 and 8.72 against NMDA and AMPA, respectively); 8‐OH‐DPAT (respective pD2 values of 9.15 and 9.00); sumatriptan (0.1 µM) was ineffective. The 5‐HT1A receptor antagonist (S)‐(+)N‐tert‐butyl‐3‐[4‐(2‐methoxyphenyl)piperazin‐1‐yl]‐2‐phenylpropionamide dihydrochloride [(+)‐WAY 100135] did not prevent the inhibition of glutamate release by 5‐HT but blocked the inhibition by 8‐OH‐DPAT of the NMDA/AMPA‐evoked cGMP responses. It is suggested that presynaptic 5‐HT1D receptors mediate inhibition directly of glutamate release and indirectly of the cGMP responses to the released glutamate; on the other hand, activation of (postsynaptic) 5‐HT1A receptors causes inhibition of the cGMP responses linked to stimulation of NMDA/AMPA receptors.
Lipid Composition in Scrapie‐Infected Mouse Brain: Prion Infection Increases the Levels of Dolichyl Phosphate and Ubiquinone
Authors: Guan Zhizhong , Söderberg Magnus , Sindelar Pavel , Prusiner Stanley B. , Kristensson Krister , Dallner Gustav
Published: July 2010

Abstract: The neutral and phospholipid composition of mouse brain infected with scrapie prions was investigated. During the later stages of this disease, the level of dolichol decreased by 30% whereas the level of dolichyl phosphate increased by 30%. In terminally ill mice, there was also a 2.5‐fold increase in both total ubiquinone and its reduced form. Furthermore, α‐tocopherol was elevated at this stage by 50%. In contrast, no changes were observed in phospholipid amount, in phospholipid composition, and in phosphatidylethanolamine plasmalogen content during the entire disease process. The fatty acid and aldehyde composition of individual phospholipids remained unaltered as well. No modifications could be detected in cholesterol content. Thus, the majority of membrane lipids in scrapie‐infected mouse brain are modified in neither quantity nor structure, but specific changes occur to a few polyisoprenoid lipids. This specificity indicates that, although prions accumulate in lysosomes, the infection process is not associated with a general membrane destruction caused by lysosomal enzyme leakage.
Changes in Phosphorylation of τ During Ischemia and Reperfusion in the Rabbit Spinal Cord
Authors: Shackelford Deborah A. , Nelson Kevin E.
Published: July 2010

Abstract: The microtubule‐associated protein τ plays an important role in the dynamics of microtubule assembly necessary for axonal growth and neurite plasticity. Ischemia disrupts the neuronal cytoskeleton both by promoting proteolysis of its components and by affecting kinase and phosphatase activities that alter its assembly. In this study the effect of ischemia and reperfusion on the expression and phosphorylation of τ was examined in a reversible model of spinal cord ischemia in rabbits. τ was found to be dephosphorylated in response to ischemia with a time course that closely matched the production of permanent paraplegia. Dephosphorylation of τ was limited to the caudal lumbar spinal cord. In a similar manner, Ca2+/calmodulin‐dependent kinase II activity was reduced only in the ischemic region. Thus, dephosphorylation of τ is an early marker of ischemia as is the rapid loss of Ca2+/calmodulin‐dependent kinase II activity, τ, however, was rephosphorylated rapidly during reperfusion at site(s) that cause a reduction in its electrophoretic mobility regardless of the neurological outcome. Alterations in phosphorylation or degradation of τ may affect microtubule stability, possibly contributing to disruption of axonal transport but also facilitating neurite plasticity in a regenerative response.
Complement Regulatory Molecules on Human Myelin and Glial Cells: Differential Expression Affects the Deposition of Activated Complement Proteins
Authors: Koski Carol L. , Estep Alden E. , Sawant‐Mane Suneeti , Shin Moon L. , Highbarger Lane , Hansch Gertrude M.
Published: July 2010

Abstract: The expression of decay‐accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b‐9 complex and C9‐C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence‐activated cell sorter analysis, and also sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody‐sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA‐treated or heat‐inactivated serum. C3b deposition was not affected by anti‐CD55 antibody. On the other hand, poly‐C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four‐ to fivefold when myelin was preincubated with anti‐CD59. Our data suggest that complement activation on myelin is down‐regulated at the step of the assembly of terminal complement complexes, including C5b‐9, due to the presence of CD59.
Characterization of Agonist‐Induced Down‐Regulation of NMDA Receptors in Cerebellar Granule Cell Cultures
Authors: Resink A. , Villa M. , Benke D. , Hidaka H. , Möhler H. , Balázs R.
Published: July 2010

Abstract: Exposure of cerebellar granule cells to NMDA in culture at 5 days in vitro, when cells are not yet vulnerable to NMDA, evoked a pronounced reduction in NMDA receptor activity, measured by NMDA‐induced 45Ca2+ influx, and counteracted the normal developmental increase in NMDA receptors. The effect was concentration and time dependent, the half‐maximal effect being reached at about 45 µM and by 4–5 h. The decrease in NMDA receptor function was accompanied by a significant reduction in the protein level of the obligatory NMDA receptor subunit (NR) NR1. Both parameters remained at a low level as long as the agonist was present. However, receptor down‐regulation was reversible, as receptor protein levels and NMDA responses were restored to control values upon NMDA removal, this process requiring protein synthesis. NMDA treatment also elicited a decrease in NR1, NR2A, and NR2B subunit messenger RNA (mRNA) levels. However, in comparison with NMDA receptor proteins, the decrease was faster, and NMDA receptor mRNA content recovered to control levels within 24 h in spite of the presence of NMDA. Concerning the mechanisms of agonist‐induced regulation of NMDA receptor expression, it seems that protein kinase C‐mediated protein phosphorylation is not involved, whereas inhibition of Ca2+/calmodulin‐dependent kinase II/IV by KN‐62 does depress NMDA receptor expression even in the absence of NMDA.
Electroconvulsive Seizure Increases the Expression of CREM (Cyclic AMP Response Element Modulator) and ICER (Inducible Cyclic AMP Early Repressor) in Rat Brain
Authors: Fitzgerald Laura Rydelek , Vaidya Vidita A. , Terwilliger Rosemarie Z. , Duman Ronald S.
Published: July 2010

Abstract: Rapid expression of ICER (inducible cyclic AMP early repressor), an inducible member of the CREM (cyclic AMP response element modulator) family of transcription factors, has been reported in neuroendocrine tissues and cell lines, but not in brain. In the present study, we demonstrate that acute electroconvulsive seizure (ECS) increases the expression of ICER in several rat brain regions. RNase protection analysis demonstrated that 1–2 h after administration of ECS, levels of mRNA for ICER and a splice variant, ICERγ, were significantly increased in hippocampus, frontal cortex, and cerebellum. It is surprising that ECS also increased levels of mRNA for several CREM isoforms that previous studies have reported were not rapidly inducible. In situ hybridization analysis confirmed these findings and demonstrated that ECS induction of ICER was most obvious in the dentate gyrus granule cell layer of hippocampus and deep layers of cerebral cortex. Induction of ICER and CREM was accompanied by increased expression of two small CRE‐binding complexes. Gel supershift analysis with CREM/ICER antisera confirmed that the inducible CRE‐binding complexes contain CREM/ICER. Induction of CREM and ICER may contribute to negative feedback regulation of gene transcription that is increased by acute seizure and activation of CREB (cyclic AMP response element‐binding protein).
Mechanisms of Injury‐Induced Calcium Entry into Peripheral Nerve Myelinated Axons: Role of Reverse Sodium‐Calcium Exchange
Authors: Lehning Ellen J. , Doshi Renu , Isaksson Norman , Stys Peter K. , LoPachin Richard M.
Published: July 2010

Abstract: To investigate the route of axonal Ca2+ entry during anoxia, electron probe x‐ray microanalysis was used to measure elemental composition of anoxic tibial nerve myelinated axons after in vitro experimental procedures that modify transaxolemmal Na+ and Ca2+ movements. Perfusion of nerve segments with zero‐Na+/Li+‐substituted medium and Na+ channel blockade by tetrodotoxin (1 µM) prevented anoxia‐induced increases in Na and Ca concentrations of axoplasm and mitochondria. Incubation with a zero‐Ca2+/EGTA perfusate impeded axonal and mitochondrial Ca accumulation during anoxia but did not affect characteristic Na and K responses. Inhibition of Na+‐Ca2+ exchange with bepridil (50 µM) reduced significantly the Ca content of anoxic axons although mitochondrial Ca remained at anoxic levels. Nifedipine (10 µM), an L‐type Ca2+ channel blocker, did not alter anoxia‐induced changes in axonal Na, Ca, and K. Exposure of normoxic control nerves to tetrodotoxin, bepridil, or nifedipine did not affect axonal elemental composition, whereas both zero‐Ca2+ and zero‐Na+ solutions altered normal elemental content characteristically and significantly. The findings of this study suggest that during anoxia, Na+ enters axons via voltage‐gated Na+ channels and that subsequent increases in axoplasmic Na+ are coupled functionally to extraaxonal Ca2+ import. Intracellular Na+‐dependent, extraaxonal Ca2+ entry is consistent with reverse operation of the axolemmal Na+‐Ca2+ exchanger, and we suggest that this mode of Ca2+ influx plays a general role in peripheral nerve axon injury.
Characterization of Insulin‐Like Growth Factor‐I and Its Receptor and Binding Proteins in Transected Nerves and Cultured Schwann Cells
Authors: Cheng Hsin‐Lin , Randolph Ann , Yee Douglas , Delafontaine Patrick , Tennekoon Gihan , Feldman Eva L.
Published: July 2010

Abstract: The insulin‐like growth factors (IGFs) are trophic factors whose growth‐promoting actions are mediated via the IGF‐I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF‐I and IGF‐I receptor after rat sciatic nerve transection. Schwann cells (SCs) were the main source of IGF‐I and IGFBP‐5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF‐I positive. In vitro, IGF‐I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF‐I receptor and IGFBP‐5. IGF‐I treatment increased the intensity of IGFBP‐5 without affecting gene expression. Des(1–3)IGF‐I, an IGF‐I analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP‐5 with SC conditioned media revealed IGF‐I protection of IGFBP‐5 from proteolysis, implying the presence of an IGFBP‐5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF‐I and SC express the IGF‐I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete IGFBPs, which in turn may increase local IGF‐I bioavailability.
Manipulation of Membrane Potential Modulates Malonate‐Induced Striatal Excitotoxicity In Vivo
Authors: Greene James G. , Greenamyre J. Timothy
Published: July 2010

Abstract: Malonate is a reversible inhibitor of succinate dehydrogenase (SDH) that produces neurotoxicity by an N‐methyl‐d‐aspartate (NMDA) receptor‐dependent mechanism. We have examined the influence of pharmacological manipulation of membrane potential on striatal malonate toxicity in rats in vivo by analysis of lesion volume. Depolarization caused by coinjection of the Na+,K+‐ATPase inhibitor ouabain or a high concentration of potassium greatly exacerbated malonate toxicity; this combined toxicity was blocked by the noncompetitive NMDA antagonist MK‐801. The toxicity of NMDA was also exacerbated by ouabain. The overt toxicity of a high dose of ouabain (1 nmol) was largely prevented by MK‐801. Coinjection of the K+ channel activator minoxidil (4 nmol) to reduce depolarization attenuated the toxicity of 1 µmol of malonate by ∼60% without affecting malonate‐induced ATP depletion. These results indicate that membrane depolarization exacerbates malonate neurotoxicity and that membrane hyperpolarization protects against malonate‐induced neuronal damage. We hypothesize that the effects of membrane potential on malonate toxicity are mediated through the NMDA receptor as a result of its combined agonist‐ and voltage‐dependent properties.
Norepinephrine Release in the Rat Pineal Gland: The Input from the Biological Clock Measured by In Vivo Microdialysis
Authors: Drijfhout W. J. , Van Der Linde A. G. , Kooi S. E. , Grol C. J. , Westerink B. H. C.
Published: July 2010

Abstract: The sympathetic innervation of the rat pineal gland was investigated, measuring the norepinephrine (NE) release by on‐line in vivo microdialysis. NE was assayed using an HPLC method with precolumn derivatization and fluorescence detection. Its high sensitivity and reliability made it very suitable to monitor the low levels of NE in the dialysates (12.5 fmol during nighttime, 3 fmol during daytime). To increase NE levels, the monoamine reuptake inhibitor cocaine was added to Ringer's solution at concentrations of 10−6 and 10−5M. This resulted in increases of neurotransmitter output of 167 and 219%, respectively, but did not change the qualitative and/or quantitative outcome of other experiments. Perfusion with 10−6M tetrodotoxin for 1 h resulted in a decrease of the NE release by >80%, whereas perfusion with the α2‐receptor antagonist yohimbine caused a twofold increase. These results indicate that the NE release in the rat pineal was of neuronal origin and regulated by a negative feedback mechanism involving inhibitory presynaptic α2‐receptors. Long‐term (i.e., 16 h) measurements are described, showing the circadian properties of NE release. A pronounced rhythm is reported, showing extremely sharp transitions between low daytime and high nighttime values. Increases and decreases are reported to occur within the duration of collecting one sample (20 min). For comparison, the rhythm of melatonin release was also recorded. The on and off switches of the sympathetic input correlated well with the circadian rhythm of melatonin release and can thus be considered as the primary clock signal, inducing the nightly production of melatonin.
Potassium Chloride Pulse Enhances Mitogen‐Activated Protein Kinase Activity in Rat Hippocampal Slices
Authors: Baron Carole , Benes Cyril , Van Tan Huynh , Fagard Remi , Roisin Marie‐Paule
Published: July 2010

Abstract: Mitogen‐activated protein (MAP) kinases have been implicated in multiple responses to extracellular stimuli. In this study we show that MAP kinase activity is enhanced after a KCI pulse. This activation correlates with an increased tyrosine phosphorylation of a 42‐kDa protein as determined by antiphosphotyrosine immunoblot. The same band is found in an anti‐MAP kinase immunoblot. Activity is enhanced within 1 min, reaches a maximum at 2 min, and returns to basal level after 10 min. A second peak of activity is observed between 12 and 30 min. The activation is completely blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), showing the involvement of the AMPA type of glutamate receptor. Partial inhibition of MAP kinase activation by 2‐amino‐5‐phosphonovalerate (APV) also shows the involvement of the NMDA receptor. Because the KCI pulse used induces long‐term potentiation (LTP) in rat hippocampal slice, we conclude that MAP kinase may be involved in neuronal transduction events leading to LTP.
Expression of Glycine Receptor Subunits in Glial Cells of the Rat Spinal Cord
Authors: Kirchhoff Frank , Mülhardt Cornel , Pastor Andrea , Becker Cord‐Michael , Kettenmann Helmut
Published: July 2010

Abstract: We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In the present study, the patch‐clamp technique was combined with the reverse transcription‐mediated PCR to analyze the glycine receptor‐subunit expression in individual glial cells of rats age 3–18 days. Using the patch‐clamp technique in the whole‐cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit‐specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all glial cell types investigated, transcripts of the α1 subunit, but not of α2 or α3 subunits, were detected. In addition, about one‐half the glial cells analyzed contained β‐subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.
Myelin Gene Expression in Immortalized Schwann Cells: Relationship to Cell Density and Proliferation
Authors: Sasagasako Naokazu , Toda Kenichi , Hollis Melissa , Quarles Richard H.
Published: July 2010

Abstract: Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions. [3H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum‐containing or serum‐free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P0 glycoprotein (P0) and myelin‐associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum‐containing medium. Furthermore, in both serum‐containing and defined media, dense cell populations expressed more MAG and P0 than sparse ones. P0 mRNA and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein‐22 (PMP‐22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP‐22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down‐regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell‐to‐cell contact.
Calcium Influx into Human Neuroblastoma Cells Induces ALZ‐50 Immunoreactivity: Involvement of Calpain‐Mediated Hydrolysis of Protein Kinase C
Authors: Shea Thomas B. , Spencer Melissa J. , Beermann Mary Lou , Cressman Corinne M. , Nixon Ralph A.
Published: July 2010

Abstract: Calcium influx into SH‐SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium‐containing medium increased ALZ‐50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ‐50 immunoreactivity. Treatment with PKC activator TPA increased ALZ‐50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in μ‐calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain‐mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady‐state levels of total τ (migrating at 46–52 kDa) but resulted in a relatively minor increase in 68‐kDa ALZ‐50‐immunoreactive τ isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ‐50 immunoreactivity, total τ levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ‐50‐immunoreactive τ isoforms were more resistant to calpain‐mediated proteolysis than were non‐ALZ‐50 reactive τ isoforms. These data therefore indicate that calpain may regulate τ levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered τ phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ‐50‐immunoreactive τ isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of τ to A68 by hyperactivation of the calpain/PKC system.
Expression of the β‐Galactoside α1,2‐Fucosyltransferase Gene Suppresses Axonal Outgrowth of Neuro2a Neuroblastoma Cells
Authors: Hitoshi Seiji , Kojima Naoya , Kusunoki Susumu , Inokuchi Jin‐ichi , Kanazawa Ichiro , Tsuji Shuichi
Published: July 2010

Abstract: The axonal outgrowth of cells of Neuro2a, a mouse neuroblastoma cell line, was suppressed on expression of the β‐galactoside α1,2‐fucosyltransferase (α1,2‐FT) gene. We recently cloned two types of rabbit α1,2‐FT, RFT‐I and RFT‐II. RFT‐I exhibits comparable kinetic properties and structural homology with human H gene α1,2‐FT, and RFT‐II shows comparable kinetic parameters with human Se gene α1,2‐FT. Neuro2a cells expressing RFT‐I (N2A‐RFT‐I) contained a large amount of fucosyl GM1 instead of GM1 and GD1a, major gangliosides in the parent Neuro2a cells, whereas Neuro2a cells expressing RFT‐II (N2A‐RFT‐II) showed a subtle change in the ganglioside pattern. N2A‐RFT‐II and parent Neuro2a cells showed axonal outgrowth in serum‐free medium on the exogenous addition of GM1, whereas N2A‐RFT‐I cells exhibited multiple neurite sprouts but not axonal outgrowth. This phenotype was fully recovered by N2A‐RFT‐I cells on the addition of d‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol and α‐l‐fucosidase to the culture medium, which resulted in pronounced reduction of fucosyl GM1 expression. These results suggested that expression of H‐type α1,2‐FT, and subsequent incorporation of fucose into glycolipids and glycoproteins, especially the formation of fucosyl GM1, modifies the response of neuronal cells to stimuli that induce axonal extension.
Increased Production of Matrix Metalloproteinases in Enriched Astrocyte and Mixed Hippocampal Cultures Treated with β‐Amyloid Peptides
Authors: Deb Suman , Gottschall Paul E.
Published: July 2010

Abstract: Growing evidence supports the notion of a functional relationship between the presence of the β‐amyloid (Aβ) peptide and the production of inflammatory mediators in and around neuritic plaques of Alzheimer's disease. Tissue remodeling enzymes that are critical in peripheral inflammatory responses are the matrix metalloproteinases (MMPs), enzymes produced by neurons and glia. Thus, it was of interest to determine whether Aβ may alter the expression of MMPs in glial and neuronal cultures. It was demonstrated that Aβ (1–40) is a potent stimulator of MMP‐9 and MMP‐2 activity in addition to inducing the expression of a lower molecular weight, unidentified gelatinase activity in mixed hippocampal and astrocyte cultures. Shorter fragments of Aβ were less effective in stimulating the production of these enzymes. The lower molecular weight activity was observed only in response to Aβ, and not after treatment with various cytokines. In addition, both cultures express MMP‐3 (stromelysin‐1) in response to Aβ peptides. These results suggest that MMPs may play a role in the development or progression of neuritic plaques, i.e., abnormal neurite outgrowth.
Adenosine Deaminase Interacts with A1 Adenosine Receptors in Pig Brain Cortical Membranes
Authors: Saura Carles , Ciruela Francisco , Casadó Vicent , Canela Enric I. , Mallol Josefa , Lluis Carmen , Franco Rafael
Published: July 2010

Abstract: Adenosine deaminase is an enzyme of purine metabolism that has largely been considered to be cytosolic. A few years ago, adenosine deaminase was reported to appear on the surface of cells. Recently, it has been demonstrated that adenosine deaminase interacts with a type II membrane protein known as either CD26 or dipeptidylpeptidase IV. In this study, by immunoprecipitation and affinity chromatography it is shown that adenosine deaminase and A1 adenosine receptors interact in pig brain cortical membranes. This is the first report in brain demonstrating an interaction between a degradative ectoenzyme and the receptor whose ligand is the enzyme substrate. By means of this interaction adenosine deaminase leads to the appearance of the high‐affinity site of the receptor, which corresponds to the receptor‐G protein complex. Thus, it seems that adenosine deaminase is necessary for coupling A1 adenosine receptors to heterotrimeric G proteins.
Ex Vivo Measurement of Brain Tissue Nitrite and Nitrate Accurately Reflects Nitric Oxide Synthase Activity In Vivo
Authors: Salter Mark , Duffy Claire , Garthwaite John , Strijbos Paul J. L. M.
Published: July 2010

Abstract: The ex vivo tissue concentration of nitrite and nitrate (NOx) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor Nω‐nitro‐l‐arginine (l‐NA) at doses that completely inhibited both central and peripheral NOS, depleted whole‐brain and CSF NOx by up to 75% but had no effect on plasma NOx. Selective inhibition of central NOS by intracerebroventricular administration of l‐NA methyl ester produced similar decreases in levels of whole‐brain NOx. A residual concentration of NOx of 10–15 µM remained in all brain regions even after complete inhibition of brain NOS. Brain NOx content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NOx was found to reflect the in vivo efficacy of several different types of NOS inhibitor: l‐NA, Nω‐monomethyl‐l‐arginine, and 7‐nitroindazole. Intraperitoneal administration of the NOS substrate l‐arginine increased brain NOx concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NOx is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo.
Alcohol Inhibits the Depolarization‐Induced Stimulation of Oxidative Phosphorylation in Synaptosomes
Authors: Li Hong Lan , Wu Shaolong , Rottenberg Hagai
Published: July 2010

Abstract: The effects of alcohol and Ca2+ transport inhibitors on depolarization‐induced stimulation of oxidative phosphorylation and free‐Ca2+ concentrations in rat synaptosomes were investigated. Glucose oxidation was stimulated by depolarization with K+ or veratridine and by the Ca2+ ionophore ionomycin. The stimulation by K+, veratridine, and ionomycin was correlated with elevation of synaptosomal free Ca2+. Depolarization‐stimulated respiration was inhibited by verapamil, Cd2+, and ruthenium red but not by diltiazem. Synaptosomal Ca2+ elevation was inhibited by verapamil but not by ruthenium red. These results indicate that the stimulation depends on elevation of mitochondrial free Ca2+. Ethanol, at pharmacological concentrations (50–200 mM), inhibited the Ca2+‐dependent stimulation of oxidative phosphorylation. This inhibition resulted, in part, from the inhibition of voltage‐gated Ca2+ channels, which inhibited the elevation of synaptosomal free Ca2+, and, in part, from the stimulation of the mitochondrial Ca2+/Na+ antiporter, which inhibited the elevation of the mitochondrial matrix free Ca2+. The inhibition by ethanol of the excitation‐induced stimulation of oxidative phosphorylation in the synapse may contribute to the depressant and narcotic effects of alcohol and enhance excitotoxicity.
Functional Expression of the Type 1 Inositol 1,4,5‐Trisphosphate Receptor Promoter‐lacZ Fusion Genes in Transgenic Mice
Authors: Furutama Daisuke , Shimoda Kouji , Yoshikawa Shingo , Miyawaki Atsushi , Furuichi Teiichi , Mikoshiba Katsuhiko
Published: July 2010

Abstract: Inositol 1,4,5‐trisphosphate receptor (IP3R) is an inositol 1,4,5‐trisphosphate (InsP3)‐gated Ca2+ release channel. Type 1 IP3R (IP3R1) is the neuronal member of the IP3R family in the CNS and is predominantly expressed in cerebellar Purkinje cells. To elucidate the molecular mechanisms responsible for coupling gene expression to neuronal InsP3/Ca2+ signaling, we have studied the structure and function of the 5′‐flanking region of the mouse IP3R1 gene. The cloned 5′‐flanking region has several sequences sharing identity with motifs for known transcriptional regulation. We have fused 5′‐flanking regions 1N from −528 to +169 and 4N from −4,187 to +169 to a β‐galactosidase gene (lacZ) as a reporter marker and have characterized their in vivo gene expression. Both 1N and 4N fusion genes functioned as a strong promoter in a neuroblastoma‐glioma hybrid cell line NG108‐15. Moreover, both 1N and 4N transgenic mouse lines carrying these 1N and 4N fusion genes showed characteristic patterns of β‐galactosidase activity in the CNS that are almost consistent with that of the endogenous IP3R1 protein, thereby suggesting that the 1N region from −528 to +169 contains sequence elements responsible for regulating gene expression in neurons and for specifying predominant expression in cerebellar Purkinje cells.
Docosahexaenoic Acid Is Required for the Survival of Rat Retinal Photoreceptors In Vitro
Authors: Rotstein Nora P. , Aveldaño Marta I. , Barrantes Francisco J. , Politi Luis E.
Published: July 2010

Abstract: The effect of docosahexaenoic acid (DHA) on neuronal survival was studied in cultured cells isolated from newborn rat retina. In vivo, the content of DHA in the retina increased nearly fourfold from days 2 to 12 after birth, whereas in retinal cells in culture it remained constant. Unlike amacrine cells, the photoreceptor cells in control cultures underwent a selective degeneration, starting at day 7, that led to their massive death by day 11. The addition of DHA at day 7 led to its active incorporation by the cultures, increasing from 6 to 21% of total fatty acids in cell lipids, and completely prevented photo‐receptor cell death. When other fatty acids were tested, both neuronal fatty acid composition and photoreceptor death were the same as in control cultures. These results indicate that DHA is specifically required for the survival of retinal photoreceptors.
2‐Deoxy‐2‐Fluoro‐d‐Glucose as a Functional Probe for NMR: The Unique Metabolism Beyond Its 6‐Phosphate
Authors: Kanazawa Y. , Yamane H. , Shinohara S. , Kuribayashi S. , Momozono Y. , Yamato Y. , Kojima M. , Masuda K.
Published: July 2010

Abstract: Epimeric conversion of 2‐deoxy‐2‐fluoro‐d‐glucose (FDG) to its 2‐epimer 2‐deoxy‐2‐fluoro‐d‐mannose (FDM) proved by 19F NMR has been shown to reflect the brain activity. To examine the feasibility of FDG as a new NMR probe for in vivo functional monitoring, we studied here the fundamental NMR properties of metabolites, spectral assignments, and reliability of NMR quantification. Metabolites confirmed in brain besides FDM‐6‐phosphate were as follows: FDG‐1‐phosphate, FDG‐1,6‐bisphosphate, FDM‐1‐phosphate, FDM‐1,6‐bisphosphate, and FDG and FDM derivatives of nucleotide diphosphate. NMR quantification of these metabolites was evaluated in comparison with the method of 18F‐labeled FDG. In the NMR functional study using FDG, where a high dose is inevitable, the dose dependence of uptake was investigated. FDG uptake in mouse brain was shown to be in the range of interpretation using the biochemical parameters of enzymes for glucose uptake as long as a dose of <200 mg/kg was used.
Expression of Glucose Transporters in Rat Brain Following Transient Focal Ischemic Injury
Authors: Urabe Takao , Hattori Nobutaka , Nagamatsu Shinya , Sawa Hiroki , Mizuno Yoshikuni
Published: July 2010

Abstract: We have investigated the serial changes in the transcription and translation of the rat glucose transporter (GLUT) 1 and 3 genes after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. Northern blot analysis and in situ hybridization study were performed to determine the chronological change and regional expression. In the ipsilateral anterior cerebral artery (ACA) cortex, GLUT1 mRNA expression was increased at 12 h (11.6‐fold) of reperfusion, and its expression was detected not only in vascular endothelial cells but also in neurons. At 48 h of reperfusion, GLUT3 mRNA expression was increased in the ipsilateral ACA (8.6‐fold) and in the contralateral MCA cortex (9.1‐fold). Immunohistochemical study failed to show GLUT1 protein synthesis in neurons in the ipsilateral ACA cortex. The immunoreactivity of GLUT3 protein was increased in neurons in ipsilateral ACA cortex and contralateral MCA cortex. Our results suggest that the expression of GLUT1 and GLUT3 is controlled differently after transient focal ischemic conditions. Furthermore, the postischemic localizations of both GLUT1 and GLUT3 expressions may be altered from the normal physiological expression pattern, which may be of importance in investigating postischemic cell function.