bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamate Inhibition of the Adrenergic‐Stimulated Production of Melatonin in Rat Pineal Gland In Vitro
Authors: Kus Laura , Handa Robert J. , McNulty John A.
Published: July 2010

Abstract:The effect ofl‐glutamate on the adrenergic‐stimulated release of melatonin in the rat pineal gland was examined using an in vitro perfusion system.l‐Glutamate by itself had no effect on melatonin secretion whereasl‐glutamate administered prior to (–)‐isoproterenol (β‐adrenergic agonist) andl‐phenylephrine (α‐adrenergic agonist) inhibited melatonin production by 42%.l‐Glutamate did not inhibit melatonin secretion when glands were stimulated with (–)‐isoproterenol alone.d‐Glutamate, as well as thel‐glutamate agonists kainate,N‐methyl‐d‐aspartate, quisqualate, andtrans‐1‐aminocyclopentane‐1, 3‐dicarboxylic acid, had no effect on the (–)‐isoproterenol‐andl‐phenylephrine‐stimulated secretion of melatonin, which suggests that the inhibitory effects of glutamate are not mediated via any of the known glutamate receptor subtypes. The possibility thatl‐glutamate may be converted to another neuroactive compound (GABA) prior to the addition of (–)‐isoproterenol andl‐phenylephrine is suggested by the observation that simultaneous administration ofl‐glutamate with (–)‐isoproterenol andl‐phenylephrine did not inhibit melatonin production.
5‐Hydroxytryptamine2and β‐Adrenergic Receptor Regulation in Rat Brain Following Chronic Treatment with Desipramine and Fluoxetine Alone and in Combination
Authors: Goodnough D. B. , Baker G. B.
Published: July 2010

Abstract:A chronic (14‐day) study was initiated to investigate the effects of combined fluoxetine (FLU) and desipramine (DMI) treatment on the densities and affinities of β‐adrenergic and 5‐hydroxytryptamine2(5‐HT2) receptors. Male Sprague‐Dawley rats were administered the following doses using osmotic minipumps: FLU, 10 mg/kg/day; DMI, 5, 10, or 15 mg/kg/day; FLU, 10 mg/kg/day, plus DMI, 5 mg/kg/day; or vehicle (distilled water). After 14 days the cortex was dissected out and used for [3H]‐ketanserin (5‐HT2) binding, [3H]CGP‐12177 (β‐adrenergic) binding, and drug level analysis. All animals receiving DMI showed significant down‐regulation of 5‐HT2receptors except those receiving FLU in combination. DMI down‐regulated β‐adrenergic receptors in a dose‐dependent manner, with significantly greater down‐regulation seen with the combination than with DMI (5 mg/kg/day) alone. This latter effect was apparently the result of greater levels of DMI in cortex with the combination than with DMI (5 mg/kg/day) alone. FLU had no effect on 5‐HT2or β‐adrenergic receptors on its own. Coadministration of FLU and DMI resulted in a doubling of levels of FLU and its demethylated metabolite, norfluoxetine (NFLU), and a tripling of DMI levels compared with values observed when FLU (10 mg/kg/day) or DMI (5 mg/kg/day) was administered alone. These results suggest that with the DMI/FLU combination (a) FLU and/or NFLU block the down‐regulation of 5‐HT2receptors caused by DMI alone, (b) an important factor determining β‐adrenergic receptor density may be the elevated DMI levels relative to those with DMI (5 mg/kg/day) alone, (c) FLU and/or NFLU inhibit the metabolism of DMI, and (d) DMI inhibits the metabolism of FLU.
Histaminergic Modulation of Hippocampal Acetylcholine Release In Vivo
Authors: Mochizuki Takatoshi , Okakura‐Mochizuki Kaori , Horii Arata , Yamamoto Yumiko , Yamatodani Atsushi
Published: July 2010

Abstract:In order to elucidate the modulatory role of the histaminergic neural system in the cholinergic neural system, the acetylcholine release from the CA1‐CA3 region in the hippocampus of anesthetized rats was studied by an in vivo microdialysis method coupled with HPLC‐electrochemical detection. The mean value for the basal acetylcholine release was 0.98 β 0.04 pmol/20 min. The acetylcholine release was increased to 172% of the basal level when an electrical stimulation at 200 μA was applied to the tuberomammillary nucleus. An administration of α‐fluoromethylhistidine (100 mg/kg i.p.) blocked the electrically evoked release of histamine both from the septal‐diagonal band complex and the hippocampus, and abolished the electrically evoked release of acetylcholine from the hippocampus. Zolantidine (5 mg/kg i.p.) attenuated the increase in the electrically stimulated acetylcholine release, but pyrilamine (5 mg/kg i.p.) did not attenuate the increase in the acetylcholine release. These drugs showed no significant effect on the basal acetylcholine release. An administration of (R)‐α‐methylhistamine (5 mg/kg i.p.) caused a decrease in the acetylcholine release to 48.7% of the basal level, whereas thioperamide (5 mg/kg i.p.) caused an increase in the acetylcholine release 60 min after the injection. These results suggest that the histaminergic system may contribute to the modulation of the activity of the septohippocampal cholinergic system, mainly through H2receptprs.
Ca2+‐Dependent Inactivation of P‐Type Calcium Channels in Nerve Terminals
Authors: Tareilus Erwin , Schoch Juergen , Breer Heinz
Published: July 2010

Abstract:Rapid Ca2+signals evoked by K+depolarization of rat cerebral cortical synaptosomes were measured by dual‐channel Ca2+spectrofluorometry coupled to a stopped‐flow device. Kinetic analysis of the signal rise phase at various extracellular Ca2+concentrations revealed that the responsible voltage‐dependent Ca2+channels, previously identified as P‐type Ca2+channels, inactivate owing to the rise in intracellular Ca2+levels. At millimolar extracellular Ca2+concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca2+, thus limiting the Ca2+signal amplitudes to 500–600 nM.A slower, probably voltage‐dependent regulation appears to be effective at lower Ca2+influx rates, leading to submaximal Ca2+signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca2+levels appears to be responsible for the different inhibition characteristics of Cd2+versus ω‐agatoxin IVa.
Carbachol, but Not Norepinephrine, NMDA, lonomycin, Ouabain, or Phorbol Myristate Acetate, Increases Inositol 1,3,4,5‐Tetrakisphosphate Accumulation in Rat Brain Cortical Slices
Authors: Myles Marvin E. , Fain John N.
Published: July 2010

Abstract:lonomycin, a Ca2+ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 mMCa2+, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5‐tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5‐trisphosphate due to 10 μMionomycin was equivalent to that seen with 1μMcarbachol. Phorbol 12‐myristate 13‐acetate or high K+(30 μM) increased inositol 1,4,5‐trisphosphate, but not inositol 1,3,4,5‐tetrakisphosphate accumulation. The stimulation of inositol 1,4,5‐trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 μMCa2+. The increase in inositol 1,3,4,5‐tetrakisphosphate accumulation in brain slices due to 1 μMcarbachol ranged from 55 to 68% of that for inositol 1,4,5‐trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5‐tris‐phosphate, but had minimal effects on inositol 1,3,4,5‐tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5‐tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase Cβ, in isolated rat brain membranes.
Extracellular N‐Acetylaspartate in the Rat Brain: In Vivo Determination of Basal Levels and Changes Evoked by High K+
Authors: Taylor Deanna L. , Davies Siân E. C. , Obrenovitch Tihomir P. , Urenjak Jutta , Richards Douglas A. , Clark John B. , Symon Lindsay
Published: July 2010

Abstract: The purpose of this study was to determine the extracellular concentrations of N‐acetylaspartate (NAA) in the rat cerebral cortex, striatum, and hippocampus of halo‐thane‐anaesthetised rats by intracerebral microdialysis, and to examine the effects of high K+‐induced local depolarisation, which provokes synchronous neurotransmitter release, cell swelling, and acid‐base changes. Basal levels of NAA in the extracellular fluid (EOF) were determined by the zero net flux method. Tissue levels of NAA in the cortex, striatum, and hippocampus were 8.4, 5.7, and 7.2 mmol/kg, respectively. The corresponding extracellular concentrations of NAA were much lower (35.1, 83.7, and 23.0 tiM). High tissue/ECF concentration ratios may suggest little release or leakage of NAA under basal conditions, and potent reuptake mechanisms for NAA in the cellular membrane of CNS cells. There was no change in ECF NAA during K+‐induced local depolarising stimuli produced in the striatum, but NAA levels consistently increased after the K+ stimuli, irrespective of whether or not Ca2+ was present in the perfusion medium. These data confirm that NAA is not a neurotransmitter and suggest strongly that NAA is not directly involved in the release and reuptake or metabolism of neuroactive compounds. The increase of NAA in the ECF immediately after K+ stimulation may reflect an involvement in brain osmoregulation and/or acid‐base homeostasis.
Time Course of Changes in Extracellular Lactate Evoked by Transient K+‐Induced Depolarisation in the Rat Striatum
Authors: Taylor D. L. , Richards D. A. , Obrenovitch T. P. , Symon L.
Published: July 2010

Abstract: The purpose of this study was to establish whether excessive lactate production associated with local application of K+ is reflected at the extracellular level during or after the K+ challenge. Changes in extracellular lactate were continuously monitored by microdialysis coupled to on‐line fluorimetric analysis. K+‐induced changes in dialysate lactate were closely related to those of the direct current potential. High K+ evoked a large and sustained negative shift of direct current potential onto which were superimposed a variable number of transient peaks of further depolarisation. The initial negative shift in direct current potential was associated with a decrease in dialysate lactate, but after each transient depolarisation, the positive shift in direct current potential indicating cell repolarisation was associated with a marked increase in extracellular lactate. When repetitive transient depolarisations occurred during a stimulus, only a small increase after each depolarisation was observed. However, recordings consistently revealed a marked and rapid increase in extracellular lactate after the K+ stimulus. These data indicate that extracellular lactate mostly increased during periods of repolarisation. This suggests strongly that lactic acid transport out of brain cells may be impaired when their transmembrane ionic gradients are disrupted.
Utilization of Uniformly Labeled 13C‐Polyunsaturated Fatty Acids in the Synthesis of Long‐Chain Fatty Acids and Cholesterol Accumulating in the Neonatal Rat Brain
Authors: Cun‐nane S. C. , Williams S. C. R. , Bell J. D. , Brookes S. , Craig K. , Iles R. A. , Crawford M. A.
Published: July 2010

Abstract: Polyunsaturated fatty acids are needed for normal neonatal brain development, but the degree of conversion of the 18‐carbon polyunsaturated fatty acid precursors consumed in the diet to their respective 20‐and 22‐carbon polyunsaturates accumulating in the brain is not well known. In the present study, in vivo 13C nuclear magnetic resonance spectroscopy was used to monitor noninvasively the brain uptake and metabolism of a mixture of uniformly 13C‐enriched 16‐and 18‐carbon polyunsaturated fatty acid methyl esters injected intragastrically into neonatal rats. In vivo NMR spectra of the rat brain at postnatal days 10 and 17 had larger fatty acid signals than in uninjected controls, but changes in levels of individual fatty acids could not be distinguished. One day after injection of the U‐13C‐polyunsaturated fatty acid mixture, 13C enrichment (measured by isotope ratio mass spectrometry) was similar in brain phospholipids, free fatty acids, free cholesterol, and brain aqueous extract; 13C enrichment remained high in the phospholipids and cholesterol for 15 days. 13C enrichment was similar in the main fatty acids of the brain within 1 day of injection but 15 days later had declined in all except arachidonic acid while continuing to increase in docosahexaenoic acid. These changes in 13C enrichment in brain fatty acids paralleled the developmental changes in brain fatty acid composition. We conclude that, in the neonatal rat brain, dietary 16‐and 18‐carbon polyunsaturates are not only elongated and desaturated but are also utilized for de novo synthesis of long‐chain saturated and monounsaturated fatty acids and cholesterol.
Lipoxygenation of Docosahexaenoic Acid by the Rat Pineal Body
Authors: Sawazaki Shigeki , Salem Norman , Kim Hee‐Yong
Published: July 2010

Abstract: Based on the inhibitor profile, production rate, and stereochemical purity of the hydroxylated products, it was demonstrated that lipoxygenation in rat brain occurs only in the pineal. Both positional and stereochemical specificities of the hydroxylation were observed only in pineal, clearly indicating that only the pineal is capable of lipoxygenating polyunsaturated fatty acids among the rat brain regions examined. Cerebral cortex also produced hydroxy products; however, they were racemic mixtures, indicating that peroxidation was responsible for their production. Rat pineal homogenate, obtained after the brain was perfused, metabolized [14C]docosahexaenoic acid ([1–14C]22:6n3) to monohydroxy derivatives, primarily by the 12‐and, to a lesser extent, by the 15‐lipoxygenase (LO) reaction. The resulting metabolites were 14(S)‐and 17(S)‐hydroxydocosahexaenoic acid (HDoHE), as determined by reversed‐phase HPLC, chiral‐phase HPLC, thermospray liquid chromatography‐mass spectrometry, and gas chromatography‐mass spectrometry. Because blood was removed by perfusion of the brain before incubation, it was clear that the observed LO activity was not due to contamination with blood cell components. The production rate of 17‐HDoHE from 22:6n3 was higher than that of 15‐hydroxyperoxy‐5,8,11,13‐eicosatetraenoic acid from 20:4n6, whereas 12‐LO activity toward these two substrates was comparable. These monohydroxy metabolites were also detected in the pineal body lipid extract using negative ion chemical ionization mass spectrometry. This is the first observation of endogenous production of hydroxylated compounds in pineal. The ratio of endogenous 15‐LO to 12‐LO products was considerably higher than that of the in vitro production from exogenous substrate. In some cases, 15‐LO products were the major LO metabolites present in the lipid extract of pineal body for both 20:4n6 and 22:6n3. Both 12‐and 15‐LO activities were recovered mainly in the microsomal plus cytosolic fraction. In addition to monohydroxy products, epoxy, hydroxy derivatives were formed from 22:6n3 by the pineal. The major isomer was identified as 12‐hydroxy‐13, 14‐epoxy‐22:5n3. Key Words: Lipoxygenation‐Docosahexaenoic acid (22:6n3)—Pineal—Rat brain—Hydroxydocosahexaenoic acid—Hepoxilin‐like compound.
Presynaptic and Postsynaptic Effects of Neuropeptide Y in the Rat Pineal Gland
Authors: Simonneaux Valérie , Ouichou AH , Craft Cheryl , Pévet Paul
Published: July 2010

Abstract:Neuropeptide Y is colocalized with noradrena‐line in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC50of 50 nM, the potassium‐evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin‐sensitive G protein‐coupled neuropeptide Y‐Y2 receptors and is independent from, but additive to, the α2‐adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC50of 5 nM, the β‐adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y‐Y1 receptors. This moderate neuropeptide Y‐induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a β‐adrenergic stimulation. On the contrary, in the presence of 1 mMascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.
Sequence of a Drosophila Ligand‐Gated Ion‐Channel Polypeptide with an Unusual Amino‐Terminal Extracellular Domain
Authors: Harvey Robert J. , Schmitt Bertram , Hermans‐Borgmeyer Irm , Gundelfinger Eckart D. , Betz Heinrich , Darlison Mark G.
Published: July 2010

Abstract: We report the isolation of a full‐length clone from a Drosophila melanogaster head cDNA library that encodes a 614‐residue polypeptide that exhibits all of the features of a ligand‐gated chloride‐channel/receptor subunit. This polypeptide, which has been named GRD (denoting that the polypeptide is a GABAA and glycine receptor‐like subunit of Drosophila), displays between 33 and 44% identity to vertebrate GABAA and glycine receptor subunits and 32–37% identity to the GABAA receptor‐like polypeptides from Drosophila and Lymnaea. It is interesting that the large amino‐terminal, presumed extracellular domain of the GRD protein contains an insertion, between the dicysteine loop and the first putative membrane‐spanning domain, of 75 amino acids that is not found in any other ligand‐gated chloride‐channel subunit. Analysis of cDNA and genomic DMA reveals that these residues are encoded by an extension of an exon that is equivalent to exon 6 of vertebrate GABAA and glycine receptor genes. The gene (named Grd) that encodes the Drosophila polypeptide has been mapped, by in situ hybridization, to position 75A on the left arm of chromosome 3.
Rapid ATP Loss Caused by Methamphetamine in the Mouse Striatum: Relationship Between Energy Impairment and Dopaminergic Neurotoxicity
Authors: Chan Piu , Di Monte Donato A. , Luo Jin‐Jun , DeLanney Louis E. , Irwin Ian , Langston J. William
Published: July 2010

Abstract:To study the relationship between energy impairment and the effects of α‐methamphetamine (METH) on dopaminergic neurons, ATP and dopamine levels were measured in the brain of C57BL/6 mice treated with either a single or four injections of METH (10 mg/kg, i.p.) at 2‐h intervals. Neither striatal ATP nor dopamine concentrations changed after a single injection of METH, but both were significantly decreased 1.5 h after the multiple‐dose regimen. The effects of METH on ATP levels appear to be selective for the striatum, as ATP concentrations were not affected in the cerebellar cortex and hippocampus after either a single or multiple injections of METH. In a second set of experiments, an intraperitoneal injection of 2‐deoxyglucose (2‐DG; 1 g/kg), an inhibitor of glucose uptake and utilization, was given 30 min before the third and fourth injections of METH. 2‐DG significantly potentiated METH‐induced striatal ATP loss at 1.5 h and dopamine depletions at 1.5 h and 1 week. These results indicate that a toxic regimen of METH selectively causes striatal energy impairment and raise the possibility that perturbations of energy metabolism play a role in METH‐induced dopaminergic neurotoxicity.
Excitotoxic Amino Acids Cause Appearance of Magnetic Resonance Spectroscopy‐Observable Zinc in Supervised Cortical Slices
Authors: Badar‐Goffer Ronnitte , Morris Peter , Thatcher Nicola , Bachelard Herman
Published: July 2010

Abstract: (1) The effects of glutamate and NMDA on the free intracellular calcium concentration ([Ca2+],) have been followed in superfused cortical slices using the 19F‐magnetic resonance indicator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N, N, N'.N'‐tetra‐acetic acid (5FBAPTA). (2) Glutamate (0.5 or 1 m/W) caused a 75–100% increase in [Ca2+], and a new resonance was attributed to zinc‐5FBAPTA, which was confirmed from its disappearance in the presence of a high‐affinity chelator of heavy metals, N, N,N',N'‐tetrakis(2‐pyridylmethyl)ethylenediamine. The appearance of zinc occurred with or just after the rise in [Ca2+], and was independent of Mg2+. (3) NMDA, N‐methyl‐dl‐aspartate, or N‐methyl‐l‐aspartate (10–200 μM) caused a slower increase in [Ca2+], and zinc was observed in some but not all experiments. When present, zinc appeared later than the increase in [Ca2+] These changes were also independent of Mg2+. (4) Decreases in both phosphocreatine and ATP were observed in all of these studies. (5) The results are discussed in terms of the proposed role of zinc as a modulator of excitotoxicity. Observations of zinc after exposure to glutamate or more slowly to NMDA, but not after depolarisation or deprivation of glucose and O2 (where increases also occur in [Da2+],), suggest that the cellular damage caused by the latter insults (depolarisation and fuel deprivation as in ischaemia) involves mechanisms not solely attributable to release of excitotoxins.
Kinesin mRNA Is Present in the Squid Giant Axon
Authors: Gioio Anthony E. , Chun Jong‐Tai , Crispino Marianna , Capano Carla Perrone , Giuditta Antonio , Kaplan Barry B.
Published: July 2010

Abstract: Recently, we reported the construction of a cDNA library encoding a heterogeneous population of polyadenylated mRNAs present in the squid giant axon. The nucleic acid sequencing of several randomly selected clones led to the identification of cDNAs encoding β‐actin and β‐tubulin, two relatively abundant axonal mRNA species. To continue characterization of this unique mRNA population, the axonal cDNA library was screened with a cDNA probe encoding the carboxy terminus of the squid kinesin heavy chain. The sequencing of several positive clones unambiguously identified axonal kinesin cDNA clones. The axonal localization of kinesin mRNA was subsequently verified by in situ hybridization histochemistry. In addition, the presence of kinesin RNA sequences in the axoplasmic polyribosome fraction was demonstrated using PCR methodology. In contrast to these findings, mRNA encoding the squid sodium channel was not detected in axoplasmic RNA, although these sequences were relatively abundant in the giant fiber lobe. Taken together, these findings demonstrate that kinesin mRNA is a component of a select group of mRNAs present in the squid giant axon, and suggest that kinesin may be synthesized locally in this model invertebrate motor neuron.
A Novel Cyclic AMP Response Element, CACTTGATC, Mediates Forskolin Induction of the Myelin Basic Protein Promoter in the Rat Schwannoma Line, D6P2T
Authors: Li Xiaobin , Wrabetz Lawrence , Cheng Yi , Kamholz John
Published: July 2010

Abstract: The rat Schwannoma cell line D6P2T constitutively expresses the mRNA encoding the major myelin protein, P0, but only expresses the mRNA encoding myelin basic protein (MBP) after exposure to forskolin or other substances that raise the levels of intracellular cyclic AMP. In this study we have investigated the molecular basis for forskolin induction of MBP transcription in D6P2T cells. We have found that a 9‐bp sequence element, CACTTGATC, located between nucleotides −85 and −77 in the MBP promoter, is necessary for forskolin induction of chloramphenicol acetyltransferase (CAT) expression after transient transfection of MBP promoter‐CAT fusion constructs into D6P2T cells. Although similar DNase I footprints, one of which is located within the above 9‐bp sequence element, are produced by nuclear extracts prepared from both forskolin‐treated and untreated cells, this same sequence can be shown to interact with a forskolin‐inducible protein complex using an electrophoretic mobility shift assay. In addition, mutation of this 9‐bp sequence abolishes both formation of this new protein‐DNA complex and forskolin‐inducible CAT expression from the heterologous SV40 promoter. Finally, we have shown that the appearance of this forskolin‐inducible protein‐DNA complex precedes that of MBP mRNA. Taken together, these data strongly support the notion that the induction of MBP transcription by forskolin in D6P2T cells is mediated by the binding of a forskolin‐inducible protein complex to the MBP promoter sequence CACTTGATC.
Neural Specificity of elav Expression: Defining a Drosophila Promoter for Directing Expression to the Nervous System
Authors: Yao Kwok‐Ming , White Kalpana
Published: July 2010

Abstract: The Drosophila melanogaster vital gene, embryonic lethal abnormal visual system (elav), is required for the postdeterminative development of the nervous system. Its gene product encodes an RNA binding protein that was found to be expressed in all neurons right after their birth. This specific, ubiquitous, and continuous pattern of neural expression has led to the increasingly popular use of ELAV protein as a neural‐specific marker. To understand the molecular basis of this neural‐specific expression, we have defined and analyzed the structure of the elav promoter. Cis‐acting sequences important for conferring the neural specificity of elav expression were identified by analyzing the reporter gene expression in transformants carrying different elav‐β‐galactosidase fusion, genes. This analysis delimits a 333‐bp region (−92 to +241) that is necessary for specifying the elav pattern of nervous system expression. A 3.5‐kb promoter fragment encompassing this region was designed for targeting gene expression specifically to the nervous system and would be a useful tool for the analysis of nervous system function.
Expression of Two Types of Nitric Oxide Synthase mRNA in Human Neuroblastoma Cell Lines
Authors: Fujisawa Hironori , Ogura Tsutomu , Kurashima Yukiko , Yokoyama Takeshi , Yamashita Junkoh , Esumi Hiroyasu
Published: July 2010

Abstract: Expression of nitric oxide synthase (NOS) was studied in nine human neuroblastoma and two human glioblastoma cell lines. Neuronal NOS (n‐NOS) mRNA of ∼10 kb was detected in four of the nine neuroblastoma cell lines by northern blot analysis using human n‐NOS cDNA as a probe. Expression of the n‐NOS mRNA was also detected in another neuroblastoma cell line in a subsequent reverse transcriptase polymerase chain reaction (RT‐PCR) study, but no n‐NOS mRNA expression was observed in the other four neuroblastoma cell lines or in the glioblastoma cell lines. The level of NOS activity correlated well with that of n‐NOS mRNA expression in neuroblastoma cell lines expressing n‐NOS mRNA. Western blot analysis showed that the n‐NOS expressed in neuroblastoma cells was a 160‐kDa protein reacted with anti‐n‐NOS antibody. By using the RT‐PCR method, a short n‐NOS (n‐NOS‐2) mRNA with a 315‐bp inframe deletion from the entire n‐NOS (n‐NOS‐1) mRNA was detected in the human neuroblastoma cells. The structural diversity of human n‐NOS mRNA was demonstrated for the first time.
α2‐Adrenoceptor‐Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP
Authors: Boehm Stefan , Huck Sigismund , Koth Gabriele , Drobny Helmut , Agneter Ernst , Singer Ernst A.
Published: July 2010

Abstract: This study explores the role of cyclic AMP in electrically evoked [3H]noradrenaline release and in the α2‐adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation‐evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine. The forskolin‐induced increase in overflow was abolished by the Rp‐diastereomer of cyclic AMP‐thioate, an antagonist at cyclic AMP‐dependent protein kinases, and 1,9‐dideoxy‐forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24‐h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin‐induced accumulation of cyclic AMP and inhibited the stimulation‐evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α2‐adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on 3H overflow were attenuated by forskolin as well as by 8‐bromo‐cyclic AMP. Effects of bromoxidine on [3H]noradrenaline release were paralleled by an inhibition of voltage‐activated Ca2+ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8‐bromo‐cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca2+ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α2‐adrenergic inhibition of noradrenaline release involves voltage‐activated Ca2+ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α2‐adrenergic reduction, apparently through a disinhibition of Ca2+ channels.
Activation of Opioid and Muscarinic Receptors Stimulates Basal Adenylyl Cyclase but Inhibits Ca2+/Calmodulin‐ and Forskolin‐Stimulated Enzyme Activities in Rat Olfactory Bulb
Authors: Olianas Maria C. , Onali Pierluigi
Published: July 2010

Abstract: In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP‐dependent and pertussis toxin‐sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca2+ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu5]Enkephalin and selective δ‐ and μ‐, but not κ‐, opioid receptors agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK‐stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu5]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca2+/CaM‐ and FSK‐stimulated enzyme activities. These results indicate that in rat olfactory bulb δ‐ and μ‐opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin‐sensitive GTP‐binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.
Temperature Sensitivity of Agonist High‐Affinity Binding Sites of Solubilized and Reconstituted D1 Dopamine Receptors
Authors: Sidhu Anita , Kimura Kazuhiro
Published: July 2010

Abstract: Solubilization of rat striatal membranes with sodium cholate, followed by reconstitution into phospholipid vesicles, leads to a 6.5‐fold increase in the agonist high‐affinity binding sites of the D1 dopamine receptor. These high‐affinity binding sites display differential sensitivity toward temperature. When reconstituted receptors were preincubated for 1 h at 0–4°C (on ice) or at 22°C (room temperature) followed by radioligand binding assays with dopamine, neither the high‐affinity values of the receptor for dopamine nor the percent receptors in the high‐affinity state (31–39%) were changed from control reconstituted receptors, which were not subject to any preincubations. At 30°C, there was a partial loss in the number of high‐affinity D1 receptors with only 25% of the total receptor population in the high‐affinity state; there was no change in the affinity values of the high‐affinity binding sites. At 37°C, there was a 40% loss in total number of D1 receptor binding sites. All the high‐affinity binding sites were lost and the remaining 60% of binding activity represented the low‐affinity binding state of the receptor. These results indicate that the high‐affinity binding sites of the reconstituted D1 dopamine receptors are uniquely sensitive to higher temperatures.
Increased Activation of L‐Type Voltage‐Dependent Calcium Channels Is Associated with Glycine Enhancement of N‐Methyl‐d‐Aspartate‐Stimulated Dopamine Release in Global Cerebral Ischemia/Reperfusion
Authors: Werling Linda L. , Hoehner Paul J. , Hurt K. Joseph , Fox Laura G. , Blanck Thomas J. J. , Rosenthal Robert E. , Fiskum Gary
Published: July 2010

Abstract: We investigated the relationships among N‐methyl‐d‐aspartate, glycine, L‐type voltage‐dependent calcium channels, and [3H]dopamine release in a canine model of global cerebral ischemia/reperfusion. The binding of [3H]PN200‐110 ([3H]isradipine) to L‐type voltage‐dependent calcium channels, that open as a consequence of N‐methyl‐d‐aspartate‐induced changes in membrane potential, was approximately doubled in striatal membranes prepared from ischemic animals relative to controls, and remained significantly elevated at 30 min and 2 h of reperfusion. These changes coincided temporally with changes in the ability of the voltage‐sensitive calcium channel blocker nitrendipine to inhibit glycine enhancement of N‐methyl‐d‐aspartate‐stimulated [3H]dopamine release in striatal slices prepared from the same animals. Compared with nonischemic controls, N‐methyl‐d‐aspartate‐stimulated [3H]dopamine release was increased in ischemic animals and remained increased throughout reperfusion up to at least 24 h. Glycine enhanced N‐methyl‐d‐aspartate‐stimulated release in all treatment groups. The enhancement of N‐methyl‐d‐aspartate‐stimulated dopamine release by glycine was reduced by the inclusion of nitrendipine in striatal slices from ischemic and 30‐min reperfused animals. These data suggest that glycine may facilitate opening of the voltage‐dependent calcium channels activated by N‐methyl‐d‐aspartate and that this facilitation is blocked by the antagonist nitrendipine.
Inhibition of Dopamine Agonist‐Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices
Authors: Undie Ashiwel S. , Friedman Eitan
Published: July 2010

Abstract: We examined the effects of cyclic AMP on dopamine receptor‐coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp‐cyclic adenosine monophosphothioate (Sp‐cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D1 receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp‐cyclic adenosine monophosphothioate (Rp‐cAMPS) dose‐dependently potentiated the SKF 38393 effect. In the presence of 200 µMRp‐cAMPS, the dose‐response curves of the dopamine D1 receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC50 values, however, were not significantly altered by protein kinase A inhibition. Neither Sp‐cAMPS nor Rp‐cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D1 receptor‐stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine‐coupled transduction pathways in at least some cells of the rat striatum.
Localization and Developmental Changes of τ Protein Kinase I/Glycogen Synthase Kinase‐3β in Rat Brain
Authors: Takahashi Miho , Tomizawa Kayoko , Kato Rika , Sato Kazuki , Uchida Tsuneko , Fujita Shinobu C. , Imahori Kazutomo
Published: July 2010

Abstract: τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase‐3β (GSK‐3β). Before elucidating a role of TPKI/GSK‐3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK‐3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.
Extension of the 2‐Deoxyglucose Method to the Fetus in Utero: Theory and Normal Values for the Cerebral Glucose Consumption in Fetal Guinea Pigs
Authors: Berger Richard , Gjedde Albert , Heck Johannes , Müller Elke , Krieglstein Josef , Jensen Arne
Published: July 2010

Abstract: Fetal cerebral metabolism changes during development. The normal fetal metabolic rate must be known to evaluate pathophysiological changes. Therefore, we determined the regional cerebral glucose consumption in the fetal guinea pig. This required the application of the 2‐deoxyglucose method to this species. We measured both the transfer coefficients of deoxyglucose and glucose between the maternal arterial plasma and the fetal brain and the lumped constant in chronically prepared undisturbed guinea pig dams using a three‐compartment model. Furthermore, the ratio between the initial clearances of deoxyglucose and glucose between the maternal arterial plasma and the fetal brain and the ratio between the phosphorylation coefficients of these substrates in the fetal brain were determined. The total cerebral glucose consumption measured by the deoxyglucose method (10 ± 1.2 µmol/100 g/min) was similar to that calculated from the glucose concentration and the phosphorylation coefficient of glucose in the cerebrum (10 ± 0.4 µmol/100 g/min). We conclude that the 2‐deoxyglucose method is applicable to the guinea pig, and we further conclude that in the fetal guinea pig cerebral glucose consumption is 10 times lower than that in the adult.
In Situ Hybridization Evidence of Differential Modulation by Pentobarbital of GABAA Receptor α1‐ and β3‐Subunit mRNAs
Authors: Tseng Yi T. , Wellman Susan E. , Ho Ing K.
Published: July 2010

Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABAA receptor α1‐ and β3‐subunit mRNA was conducted using synthetic 3′‐ end 35S‐dATP‐labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α1‐subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β3‐subunit mRNA were not affected. Dramatically increased levels of GABAA receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α1 subunit and in cerebral cortex only for the β3‐subunit. These data provide further support to the structural and pharmacological GABAA receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up‐ and down‐regulation observed in receptor ligand binding studies.
Novel Inactive and Distinctively Glycosylated Forms of Butyrylcholinesterase from Chicken Serum
Authors: Weikert Thomas , Ebert Christoph , Rasched Ihab , Layer Paul G.
Published: July 2010

Abstract: Three different homologues of butyrylcholinesterase (BChE) with 75‐, 62‐, and 54‐kDa subunit size are isolated from adult chicken serum, and all show very low or zero enzyme activity. Although the active BChE from serum with a subunit size of 81 kDa forms tetramers, the 75‐kDa protein is isolated as a dimer. The homology of the 75‐kDa protein with active BChE is shown by immunoreactivity with BChE‐specific monoclonal antibodies, by coisolation with the active BChE, and by their identical first six N‐terminal amino acids. By deglycosylation of these proteins and by their differential lectin binding, we show that the active BChE is an N‐glycosylated protein of the triantennary type, whereas the inactive 75‐kDa protein is O‐glycosylated. These data show for the first time the existence of (1) multiple inactive forms of BChE, (2) secreted inactive cholinesterases, because they are found in serum, and (3) an O‐glycosylated cholinesterase. Because cholinesterases can regulate neurite growth in vitro by a nonenzymatic mechanism, these data strongly support that both inactive and active forms of BChE may be involved in noncholinergic communication, possibly depending on particular glycosylation patterns.
Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain
Authors: Hashimoto Yoshihide , Furukawa Shoei , Omae Fumio , Miyama Yukio , Hayashi Kyozo
Published: July 2010

Abstract: Effects of thyroxine (T4) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T4 caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2‐day‐old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T4 was effective on the post‐natal rats only up to day 11. These results suggest that T4 plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region‐ and/or stage‐specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF‐responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T4 suggest that the effects of T4 on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T4.
Up‐Regulation of Carbonic Anhydrase Isozyme IV in CNS Myelin of Mice Genetically Deficient in Carbonic Anhydrase II
Authors: Brion Luc P. , Suarez Carlos , Zhang Hong , Cammer Wendy
Published: July 2010

Abstract: Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid‐base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane‐associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up‐regulates CNS CA IV. We speculate that this up‐regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.
Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion
Authors: Zhang Kejia , Filbin Marie T.
Published: July 2010

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)‐like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P0 protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig‐like domain, stabilized by a putative Cys21‐Cys98 disulfide bond. To test if this bond is indeed necessary to the adhesive function of P0, the nucleotides in the P0 cDNA coding for Cys21 were altered to code for an alanine. The mutated P0 cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P0 protein was characterized, and the adhesiveness of Cys21‐mutated P0‐expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P0‐expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys21‐mutated P0 were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P0 protein, when mutated at Cys21, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.
NMDA and Nitric Oxide Increase Microtubule‐Associated Protein 2 Gene Expression in Hippocampal Granule Cells
Authors: Johnston H. M. , Morris B. J.
Published: July 2010

Abstract: Microtubule‐associated protein 2 (MAP2), a component of the neuronal cytoskeleton, has attracted attention as a possible cellular substrate linking hippocampal N‐methyl‐d‐aspartate (NMDA) receptor stimulation to alterations in cellular morphology. We show here that microinjection of NMDA, 8‐bromo‐cyclic GMP, or sin‐1 molsidomine (which spontaneously releases nitric oxide), onto the molecular layer of the hippocampal dentate gyrus, increased the levels of MAP2 mRNA in the affected granule cells. No changes were observed in the levels of mRNAs encoding several other cytoskeletal components. This shows that hippocampal NMDA receptor stimulation can potentially initiate a long‐term alteration in dendritic structure by affecting MAP2 gene expression and provides the first evidence that nitric oxide release in vivo, acting through cyclic GMP‐dependent protein kinase, can cause long‐term changes in neuronal function by modulating gene expression.