bluuebunny/crossref_metadata_2025 huggingface dataset:
Presynaptic Noradrenergic Regulation of Glutamate Inputs to Hypothalamic Magnocellular Neurones
Authors: Boudaba C. , Di S. , Tasker J. G.
Published: July 2010

Glutamate and norepinephrine transmitter systems play critical roles in the synaptic control of hypothalamic magnocellular neurones. We recently reported on a norepinephrine‐sensitive glutamate circuit within the paraventricular nucleus (PVN) that projects to magnocellular neurones. Here, we present evidence for norepinephrine regulation of glutamate release in the PVN and supraoptic nucleus (SON) via actions on presynaptic terminals. Whole‐cell synaptic currents were recorded in magnocellular neurones of the SON and PVN in an acute slice preparation. Bath application of norepinephrine (100 µm) caused a robust, reversible increase in the frequency of spontaneous glutamatergic excitatory postsynaptic currents in 100% of SON neurones (246%) and in 88% of PVN magnocellular neurones (259%). The norepinephrine‐induced increase in glutamate release was mediated by activation of both presynaptic α1 receptors and α2 receptors, but the α1‐receptor component was the predominant component of the response. The presynaptic actions of norepinephrine were predominantly, although not completely, resistant to blockade of Na‐dependent spikes, implicating a presynaptic terminal locus of action. Interestingly, the spike‐dependent component of the response was greater in PVN than in SON magnocellular neurones. This robust presynaptic facilitation of glutamate release by norepinephrine, combined with the known excitatory postsynaptic actions of norepinephrine, activational effects on local glutamate circuits, and inhibitory effects on γ‐aminobutyric acid release, indicate a strong excitatory role of norepinephrine in the regulation of oxytocin and vasopressin release during physiological stimulation.
[3H] CGP 39653 Binding to the Agonist Site of theN‐Methyl‐ D‐Aspartate Receptor Is Modulated by Mg2+and Polyamines Independently of the Arcaine‐Sensitive Polyamine Site
Authors: Reynolds Ian J.
Published: July 2010

Abstract:This study investigated the binding of [3H] CGP 39653, a novel high‐affinity antagonist of theN‐methyl‐D‐ aspartate (NMDA) recognition site of the NMDA receptor complex. [3H] CGP 39653 bound to the NMDA receptor in well washed rat brain membranes with an affinity of about 15 nM.Other NMDA site drugs inhibited [3H] CGP 39653 binding with the following order of potency: DL‐(tetrazol‐5‐ yl)glycine > glutamate > CGS 19755 > DL‐2‐amino‐5‐ phosphonovalerate (DL‐AP5) > NMDA. Glycine and 5, 7‐ dichlorokynurenate partially inhibited binding. The poly‐amines spermine and spermidine increased [3H] CGP 39653 binding (EC50values of 10 and 22 μM, respectively). This effect was mimicked by arcaine, 1, 5‐diethylaminopiperidine, diaminodecane, diethylenetriamine, and Mg2+. The increase in [3H] CGP 39653 was a result of an increased affinity of the binding site for the ligand with very little effect on binding site density. Spermine and Mg2+also increased the affinity of the antagonists DL‐AP5 and CGS 19755, but had only minor effects on the affinity of glutamate and NMDA. Arcaine did not reverse the enhancement of [3H] CGP 39653 binding by spermine, spermidine, or Mg2+. Channel‐blocking dissociative anesthetics, including dizocilpine and ketamine, did not alter basal or Mg2+‐stimulated [3H] CGP 39653 binding. Spermine did not alter either the enhancement of [3H]‐ dizocilpine by glutamate or the inhibition of [3H]dizocilpine by DL‐AP5 or CGS 19755. These studies show that poly‐amines and divalent cations selectively enhance the affinity of antagonists for the agonist binding site on the NMDA receptor complex. However, this effect is mediated by a site independent of the primary polyamine site defined using [3H] dizocilpine binding.
Spontaneous Release of Acetylcholine from TorpedoSynaptosomes: Effect of Cetiedil and Its Analogue MR 16728
Authors: Moulian N. , Gaudry‐Talarmain Y. Morot , Israël M.
Published: July 2010

Abstract: The effects of cetiedil and its analogue MR16728 were examined on spontaneous acetylcholine release measured with a chemiluminescent assay using choline oxidase in a synaptosomal suspension obtained from Torpedo marmorata electric organ. Evoked acetylcholine release is inhibited by cetiedil, whereas this drug enhances spontaneous extracellular Ca2+‐independent acetylcholine release (up to 340%). This effect was examined as a function of cetiedil concentration and incubation time. On the other hand, the analogue MR16728, which enhances A23187‐evoked acetylcholine release, also enhances spontaneous Ca2+‐independent acetylcholine release. Cetiedil and MR16728 effects on spontaneous acetylcholine release were also examined in the presence of Ca2+. Addition of Ca2+ enhanced spontaneous acetylcholine release by 75%, and cetiedil and MR16728 stimulation was maintained but with different levels of enhancement. Thus, these results show that the processes responsible for evoked and spontaneous acetylcholine release are sensitive but in different ways to drugs of the cetiedil family.
Thyrotropin‐Releasing Hormone Release Is Enhanced in Hippocampal Slices After Electroconvulsive Shock
Authors: Knoblach S. M. , Kubek M. J.
Published: July 2010

Abstract: Hippocampal thyrotropin‐releasing hormone (TRH) release was examined after seizures were induced by electroconvulsive shock (ECS). Rat hippocampal slices taken 12, 24, or 48 h after 3 days of alternate‐day ECS treatment or sham‐ECS treatment were stimulated with potassium with or without calcium in a superfusion system containing in‐line charcoal adsorbent to concentrate TRH. Released TRH and tissue TRH were measured by radioimmunoassay. The TRH content of hippocampal slices was increased fivefold over sham‐ECS levels 12, 24, and 48 h after ECS, but this was not associated with an increase in basal TRH release. Potassium‐stimulated TRH release was significantly elevated over basal release 12, 24, and 48 h after ECS. Potassium‐stimulated calcium‐dependent TRH release increased linearly after ECS, reaching its highest level 48 h after seizure. Thus, although enhanced calcium‐dependent TRH release was associated with elevated tissue levels, this relationship was not proportional in that tissue TRH was elevated to the same extent at all times after ECS, whereas potassium‐evoked calcium‐dependent TRH release increased gradually over time after seizure. These results suggest that postictal elevations in TRH are associated with an enhanced capacity for release that develops as a result of a time‐dependent shift of TRH from a storage compartment to a readily releasable pool. The observed elevation in stimulated TRH release may be relevant to seizure‐induced modulation of TRH receptors in vivo.
Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood‐Brain Barrier by Intracarotid Infusion of Protamine Sulfate
Authors: Westergren Irena , Nystrom Britta , Hamberger Anders , Nordborg Claes , Johansson Barbro B.
Published: July 2010

Abstract: This article evaluates the influence of an opening of the blood‐brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood‐CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.
Tyrosination State of α‐Tubulin in Regenerating Peripheral Nerve
Authors: Mullins Fraser H. , Hargreaves Alan J. , Li Jia‐Yi , Eahlström Annica , McLean W. Graham
Published: July 2010

Abstract: Certain modifications of the neuronal cytoskeleton that are associated with development also occur during regeneration of adult mammalian peripheral nerve. The aim of the present study was to examine one such modification, the tyrosination of a‐tubulin. Adult rats were anaesthetized and the left or right sciatic nerve randomly selected and crushed to induce regeneration. In certain instances nerves were crushed then ligatured about the crush, to prevent regeneration. Five days later the rats were killed and the regenerating (or ligatured) and the contralateral (control) nerves were removed. Quantitative immunoblotting of nerve homogenates with antibodies that recognize tyrosinated a‐tubulin and total a‐tubulin revealed a significant increase (p < 0.01) in the proportion of a‐tubulin that was tyrosinated in nerve pieces distal (peripheral) to a nerve crush compared with nerve pieces proximal (central) to a nerve crush and to uncrushed nerve. No such difference occurred in ligatured (crushed but nonregenerating) nerve, implying that the increase was related to the presence of regenerating fibres; nor was there any gradient in tyrosination of α‐tubulin in control nerves. This effect was confirmed by cytofluorimetric scanning and fluorescence confocal laser scanning microscopy of fixed sections of control and regenerating nerve, stained with antibodies directed against tyrosinated a‐tubulin. When nerves were separated into fractions containing assembled and nonassembled tubulin, a significant (p < 0.01) increase was found in the proportion of tyrosinated α‐tubulin in the nonassembled tubulin fraction in nerve pieces containing regenerating fibres. This occurred in the absence of a change in the proportion of assembled and nonassembled tubulin. Measurements of tubulin:tyrosine ligase activity, by incorporation of [3H] tyrosine into endogenous nerve tubulin in vitro, indicated a decrease in tyrosine incorporation into tubulin from nerve pieces distal, compared with those proximal to a nerve crush. There was no such difference in ligatured nerves. It is proposed that the increased amount of tyrosinated a‐tubulin is related to an alteration in assembly rate of microtubules required for neurite outgrowth and that the apparent decrease in the tubulin:tyrosine ligase activity in vitro reflects the increased tyrosination in vivo.
Developmental Expression of GLUT1 and GLUT3 Glucose Transporters in Rat Brain
Authors: Vannucci Susan J.
Published: July 2010

Abstract: Two glucose transport proteins, GLUT1 and GLUT3, have been detected in brain. GLUT1 is concentrated in the endothelial cells of the blood‐brain barrier and may be present in neurons and glia; GLUT3 is probably the major neuronal glucose transporter. Of the few studies of glucose transport in the immature brain, none has quantified GLUTS. This study used membrane isolation and immunoblotting techniques to examine the developmental expression of GLUT1 and GLUT3 in four forebrain regions, cerebral microvessels, and choroid plexus, from rats 1–30 days postnatally as compared with adults. The GLUT1 level in whole brain samples was low for 14 days, doubled by 21 days, and doubled again to attain adult levels by 30 days; there was no regional variation. The GLUT3 level in these samples was low during the first postnatal week, increased steadily to adult levels by 21–30 days, and demonstrated regional specificity. The concentration of GLUT1 in microvessels increased steadily after the first postnatal week; the GLUT1 level in choroid plexus was high at birth, decreased at 1 week, and then returned to near fetal levels. GLUT3 was not found in microvessels or choroid plexus. This study indicates that both GLUT1 and GLUT3 are developmentally regulated in rat brain: GLUT1 appears to relate to the nutrient supply and overall growth of the brain, whereas GLUT3 more closely relates to functional activity and neuronal maturation.
Long‐Chain os‐Isoprenyltransferase Activity Is Induced Early in the Developmental Program for Protein N‐Glycosylation in Embryonic Rat Brain Cells
Authors: Crick Dean C. , Waechter Charles J.
Published: July 2010

A large developmental increase in Glc3Man9‐ GlcNAc2‐P‐P‐dolichol (Oligo‐P‐P‐Dol) synthesis and protein W‐glycosylation in primary cultures of embryonic rat brain cells has been reported previously. In vitro enzyme studies and metabolic labeling experiments now show that there is a coordinate induction of long‐chain c/s‐iso‐ prenyltransferase (IPTase) activity, an activity required for the chain‐elongation stage of dolichyl monophosphate (Dol‐P) biosynthesis de novo, and Oligo‐P‐P‐Dol biosynthesis in embryonic rat brain. Different developmental patterns were observed for IPTase and |8‐hydroxy‐/3‐methyl‐ glutaryl‐CoA (HMG‐CoA) reductase activity as well as Dol‐ P and cholesterol biosynthesis, indicating that these pathways are regulated independently in rat brain. Three separate experimental approaches provide evidence that the amount of Dol‐P available in the rough endoplasmic reticulum (RER) is a rate‐limiting factor in the expression of the lipid intermediate pathway. First, metabolic labeling experiments show that the biosynthesis of Dol‐P is induced at the same time or just prior to the induction of Oligo‐P‐P‐Dol biosynthesis. Second, the time of induction and rate of Oligo‐P‐P‐Dol synthesis are accelerated when Dol‐P is supplemented in the culture medium. Third, in vitro assays of mannosylphosphoryldolichol synthase and A/‐acetylglucosaminylpyrophosphoryldolichol synthase indicate that there are only minor increases in the levels of these enzymes during development, but the amount of endogenous Dol‐P in the RER that is accessible to the glycosyltransferases increases when IPTase activity is induced. In summary, the current studies with embryonic rat brain cells document the coordinate induction of IPTase activity and Oligo‐P‐P‐Dol synthesis, support the hypothesis that the availability of Dol‐P in the RER is one rate‐limiting factor in Oligo‐P‐P‐Dol synthesis, and strongly suggest that increases in IPTase activity and the rate of de novo Dol‐P biosynthesis enhance the capacity of embryonic rat brain cells for lipid intermediate synthesis early in the developmental program for N‐linked glycoprotein biosynthesis.
Axonal Membrane‐Skeletal Protein A60: Association with a Brain Spectrin‐Binding Activity and Entry into Cerebellar Axons at a Stage After the Initiation of Axonal Growth
Authors: Hayes Nandini V. L. , Baines Anthony J.
Published: July 2010

Abstract: A60 is a 60‐kDa component of the axonal cortical cytoskeleton in CNS neurones. It appears to be neurone specific and is tightly bound to brain membranes. In this study the cytoskeletal activities and developmental expression of A60 in rat cerebellum have been examined using the monoclonal antibody DR1. A60 in a partially purified soluble extract of brain membranes interacts selectively with brain but not erythrocyte spectrin. Because erythrocyte spectrin is more closely related to the dendritic form of spectrin than the axonal form, this raises the possibility that AGO localises in axons by interaction with the axonal form of spectrin only. A60 is not found in rat cerebellum before the day of birth. However, during postnatal development of the cerebellum (days 1–13) DR1 reactivity appears progressively. On postnatal day 1, a small population of cells in the mantle layer (presumptive Purkinje cells) is DR1 positive. There is no DR1 reactivity found in Purkinje cell axons during their initial phase of growth. By postnatal day 7, Purkinje cell bodies, initial dendritic segments, and the cerebellar white matter are all positive. This pattern of labelling is strengthened up until postnatal day 13. By contrast, in adult rat cerebellum, the location of A60 has changed so that it is most concentrated in axons, and dendritic staining is lost. These data indicate that A60 is a spectrin‐binding component of the adult axonal membrane skeleton, the presence of which is only required in axons after the initial phase of growth.
Rapid Communication:cis‐Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor
Authors: Huff Robin A. , Abou‐Donia Mohamed B.
Published: July 2010

Abstract:cis‐Methyldioxolane (CD) is a muscarinic receptor agonist. [3H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [3H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [3H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [3H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty‐eight percent of Hm2 receptors bound [3H] CD with aKDof 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5′‐O‐(3‐thiotriphosphate), indicating that the Hm2 receptors labeled by [3H] CD are those that are associated with GDP‐bound G protein. Binding of [3H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [3H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [3H] CD should aid research in these areas.
Ontogeny of Kainate‐Induced Gene Expression in Rat Hippocampus
Authors: Pennypacker K. R. , McMillian M. K. , Douglass J. , Hong J. S.
Published: July 2010

Abstract: The ontogeny of kainate induction of AP‐1 mRNAs, proteins, and DNA binding activities was examined in the rat hippocampus. In addition, kainate induction of preproenkephalin and preprodynorphin mRNAs was examined; these genes have been shown to be induced by kainate and have been suggested to be targets of AP‐1 regulation in adult rat hippocampus. Despite producing seizures at postnatal day (P) 7, kainate failed to induce AP‐1 or opiate gene expression and did not increase AP‐1 DNA binding activity at this age. Basal levels of AP‐1 and opiate mRNAs were low in P7 hippocampus. Basal levels of c‐jun protein and AP‐1 DNA binding activity were elevated in the P7 hippocampus, to values greater than induced levels in adult hippocampus. Furthermore, AP‐1 DNA binding in P7 hippocampal nuclear extract was unaffected by antibodies against fos‐related antigens, in contrast to hippocampal extracts from the older rats examined. At P14, induction of AP‐1 and preproenkephalin (but not preprodynorphin) mRNAs was observed with kainate treatment, but the time course for inductions was delayed relative to kainate inductions in the adult hippocampus. At P21, responses to kainate were similar to the adult response. Unlike in adult hippocampus, seizure activity caused by kainate treatment does not increase the transcription factor and opioid peptide gene expression in the hippocampi of P7 rats.
Calcium‐Independent Release of Acetylcholine from Stable Cell Lines Expressing Mouse Choline Acetyltransferase cDNA
Authors: Misawa Hidemi , Takahashi Ryosuke , Deguchi Takeo
Published: July 2010

Abstract: Stably transfected cells expressing mouse choline acetyltransferase (ChAT) cDNA were established, and the synthesis and release of acetylcholine (ACh) were examined. A cDNA clone coding for mouse ChAT was inserted into an expression vector (pEF321) containing a promoter for human elongation factor 1α to construct pEFmChAT. Neuronal (NG108‐15, NS20Y, N1E115, and Neuro2A) and nonneuronal cell lines (L cells and NIH3T3) were transfected with pEFmChAT, and the cell lines that stably expressed high ChAT activity were selected. These cells expressed the 66‐kDa ChAT protein and accumulated ACh mostly in the cytosol. The concentration of intracellular ACh in the cells increased upon raising the choline level in the medium. The cells continuously released ACh in a Ca2+‐independent fashion. Neither high K+ nor calcium ionophore stimulated release of ACh from the cells.
Muscarinic Receptor‐Mediated Increase in Extracellular Inositol 1,4,5‐Trisphosphate Levels in the Rat Hippocampus: An In Vivo Microdialysis Study
Authors: Minisclou C. , Rouquier L. , Benavides J. , Scatton B. , Claustre Y.
Published: July 2010

Abstract: The extracellular concentration of inositol 1,4,5‐trisphosphate (IP3) has been monitored in the ventral hippocampus of the anesthetized rat by using a microdialysis technique coupled to a radioreceptor assay. Three hours after the implantation of the cannula, basal extracellular concentration of IP3 (corrected for a 9% recovery) was 71 nM (0.39 pmol/60‐µl fraction) and remained stable for at least 5 h. Local infusion of carbachol for 60 min caused a significant concentration‐related increase in extracellular IP3 levels (0, 24, and 57% at 1, 50, and 100 µM, respectively). Acetylcholine (100 µM) and muscarine (100 µM) increased IP3 outflow by 40 and 42%, respectively. The effect of carbachol was fully prevented by coinfusion of 10 µM pirenzepine and reduced by 1 µM tetrodotoxin indicating that the carbachol response is mediated by neuronal muscarinic receptors. These data demonstrate the feasibility of using microdialysis and a radioreceptor assay to measure IP3 in the extracellular space. This approach could prove useful for the study of the in vivo operation of muscarinic and, by extension, a number of receptors coupled to phosphoinositide turnover.
Zinc Inhibition of t‐[3H]Butylbicycloorthobenzoate Binding to the GABAA Receptor Complex
Authors: Kume Akito , Sakurai Sharin Y. , Albin Roger L.
Published: July 2010

Abstract: The effect of Zn2+ on t‐[3H]butylbicycloorthobenzoate ([3H]TBOB) binding to the GABAA receptor complex was studied autoradiographically in rat brain. Zn2+ inhibited [3H]TBOB binding in a dose‐dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn2+ had regional heterogeneity; regions showing the greatest inhibition of [3H]TBOB binding were cortical laminae I–III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [3H]TBOB binding included cortical laminae V–VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn2+ and those of other GABAA ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [3H]TBOB binding seemed to be additive. Ni2+, Cd2+, and Cu2+ also inhibited [3H]TBOB binding with a regional heterogeneity similar to that produced by Zn2+. These results are consistent with Zn2+ acting at the previously detected recognition site on the GABAA receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn2+ effect may reflect differential regional distribution of GABAA receptor subtypes among brain regions. Other divalent cations probably act at the Zn2+ binding site.
Effect of Pargyline on Brain N‐tele‐Methylhistamine in Portocaval‐Shunted Rats: Relation to Amine Neurotransmitters
Authors: Fogel W. A. , Tuomisto L. , Sasiak K. , Rokicki W. , Rokicki M. , MacDonald E. , Maslinski C.
Published: July 2010

Abstract: HPLC determination of histamine, serotonin, dopamine, and noradrenaline in the brain tissue of rats with portocaval anastomoses (PCA) has revealed a selective increase in histamine concentration. In the posterior hypothalamus, the steady‐state level of the amine metabolites showed an inverse pattern; N‐tele‐methylhistamine(t‐MeHA), as estimated by gas chromatography‐mass spectrometry, was not changed significantly by portocaval shunting, whereas 5‐hydroxyindoleacetic acid (5‐HIAA) and homovanillic acid were more than doubled. Interestingly, the net increase in t‐MeHA concentration in response to pargyline (80 mg/kg i.p.) was almost the same for PCA and sham‐operated rats. This implies that the great enhancement of the histamine level in this area might be a consequence of the persistent stimulation of its synthesis and the unchanged activity of histaminergic neurons. In the rest of the brain, on the other hand, the steady‐state level of t‐MeHA was higher after PCA (3.8‐fold), as were the levels of 5‐HIAA and homovanillic acid. Surprisingly, t‐MeHA remained unchanged after monoamine oxidase blockade. Of the pargyline‐induced alterations in the concentrations of indoles and catechols, the most pronounced were those in the serotonin level; serotonin was elevated more than twofold in hypothalamus and more than 12‐fold in the rest of the brain, with a concomitant 80% decrease in 5‐HIAA. The dopamine and, to a much smaller extent, noradrenaline levels were also increased, and the levels of homovanillic acid and 3,4‐dihydroxyphenylacetic acid fell below the detection limit. The study suggests that at least two different mechanisms operate in the brains of PCA rats to counteract the excessive synthesis of neuromediators, e.g., increased deposition and increased metabolism.
Region‐Selective Decreases in Densities of [3H]Tryptamine Binding Sites in Autopsied Brain Tissue from Cirrhotic Patients with Hepatic Encephalopathy
Authors: Mousseau Darrell D. , Layrargues Gilles Pomier , Butterworth Roger F.
Published: July 2010

Abstract: The distribution of [3H]tryptamine binding sites, in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy (HE) and an equal number of age‐matched control subjects free from hepatic, neurological, or psychiatric disorder, was investigated. Scatchard analysis demonstrated a heterogeneous distribution for this binding site, with the highest density being observed in hippocampus ≫ frontal cortex = caudate nucleus > temporal cortex = cerebellum. When comparing [3H]‐tryptamine binding site densities in control brain tissue with that in brain tissue from patients with HE, significant decreases in densities were observed in the frontal cortex (by 56%, p < 0.001), hippocampus (by 43%, p < 0.001), and caudate nucleus (by 41%, p < 0.01) of the HE group. Binding site affinities were within normal limits. The findings of decreased densities of [3H]tryptamine binding sites taken in conjunction with previous reports of increased CSF and brain tryptamine concentrations in HE suggest a pathogenic role for this neuroactive amine in HE resulting from chronic liver failure.
Transport of Zinc‐65 at the Blood‐Brain Barrier During Short Cerebrovascular Perfusion in the Rat: Its Enhancement by Histidine
Authors: Buxani‐Rice S. , Ueda F. , Bradbury M. W. B.
Published: July 2010

Abstract: Zinc‐65 transport into different regions of rat brain has been measured during short vascular perfusion of one cerebral hemisphere with an oxygenated HEPES‐containing physiological saline at pH 7.40. The [Zn2+] was buffered with either bovine serum albumin or histidine. In each case uptake was linear with time up to 90 s. 65Zn flux into brain in the presence of albumin followed Michaelis‐Menten kinetics and for parietal cortex had a Km of 16 nM and a Vmax of 44 nmol/kg/min. Increasing concentrations of l‐histidine enhanced 65Zn flux into brain at [Zn2+] values between 1 and 1,000 nM. The combined effect of [histidine] and [Zn2+] was best accounted for by a function of [ZnHis+], i.e., flux = 64.4 · [ZnHis+]/(390 + [ZnHis+]) + 0.00378 · [ZnHis+], with concentrations being nanomolar. d‐Histidine had an influence similar to that of l‐histidine. 65Zn flux in the presence of 100 µMl‐histidine was not affected by either 500 µMl‐arginine or 500 µMl‐phenylalanine. The results indicate specific transport of Zn2+ across the plasma membranes of brain endothelium. The enhancement due to histidine has been attributed to diffusion of ZnHis+ across unstirred layers “ferrying” zinc to and from transport sites.
The Peripheral‐Type Benzodiazepine Receptor Is Present in Astrocytes but Is Not a Primary Site of Action for Convulsants/Anticonvulsants
Authors: Moynagh Paul N. , Schousboe Arne , Williams D. Clive
Published: July 2010

Abstract: High‐affinity binding sites for [3H]PK 11195 and [3H]Ro 5‐4864 with the properties of the peripheral‐type benzodiazepine receptor were detected in primary cultures of both mouse neocortical and cerebellar astrocytes. The binding sites were enriched in mitochondrial fractions on differential centrifugation. An 18‐kDa polypeptide was specifically photolabelled in cerebellar astrocytes by [3H]‐PK 14105, a photolabel for the peripheral‐type benzodiazepine receptor. However, this polypeptide did not show any reactivity with an antiserum previously raised against the corresponding polypeptide from rat adrenal gland. Various anticonvulsant and convulsant agents were tested for their ability and potency at inhibiting [3H]Ro 5‐4864 binding to neocortical astrocytes. Many of these compounds, previously reported to be inhibitors of diazepam binding to neocortical astrocytes, proved ineffective in this study. No correlation was observed between convulsant/anticonvulsant potency and ability to inhibit [3H]Ro 5‐4864 binding to the peripheral‐type benzodiazepine receptor in these cells. Thus, whereas some convulsants and anticonvulsants might interact with this astrocytic receptor, such a system has no validity as a general screening method for these agents.
Elevated Tyrosine Hydroxylase in the Locus Coeruleus of Suicide Victims
Authors: Ordway Gregory A. , Smith Karen Streator , Haycock John W.
Published: July 2010

Abstract: The amounts of tyrosine hydroxylase protein in locus coeruleus from nine pairs of antidepressant‐free suicide victims and age‐matched, sudden‐death control cases were determined by quantitative blot immunolabeling of cryostat‐cut sections from the caudal portion of the nucleus. In each of the nine age‐matched pairs, the concentration of tyrosine hydroxylase was greater in the sample from the suicide victim, with values ranging from 108 to 172% of the matched control value (\‐x = 136%). By contrast, there were no differences in the concentrations of neuron‐specific enolase protein in the same set of samples. Similarly, the number of neuromelanin‐containing cells, counted in sections of locus coeruleus adjacent to those taken for blot immunolabeling analyses, did not differ between the two groups. These data indicate that locus coeruleus neurons from suicide victims contain higher than normal concentrations of tyrosine hydroxylase, thus raising the possibility that the expression of tyrosine hydroxylase in locus coeruleus may be relevant in the pathophysiology of suicide.
Phospholipase A2 Stimulation by Methyl Mercury in Neuron Culture
Authors: Verity M. A. , Sarafian T. , Pacifici E. H. K. , Sevanian A.
Published: July 2010

Abstract: In primary prelabeled cultures of cerebellar granule cells, methyl mercury (MeHg) induced a concentration‐ and time‐dependent release of [3H]arachidonic acid. MeHg‐induced [3H]arachidonate release was partially dependent on the extracellular Ca2+ concentration. MeHg at 10–20 µM also stimulated basal 45Ca2+ uptake after 20 min of incubation at 37°C, and at 10 µM inhibited K+ depolarization‐stimulated uptake. MeHg stimulated [3H]arachidonate uptake, but had no effect on the rate of phospholipid reacylation. Phospholipase A2 (PLA2) activation preceded cytotoxicity, but at higher concentrations of MeHg such dissociation was not evident. Inhibition of MeHg‐induced PLA2 activation by 100 µM mepacrine failed to modify cytotoxicity. MeHg‐induced lipoperoxidation, measured as the production of thiobarbituric acid‐reacting products, was inhibited by α‐tocopherol without inhibition of [3H]arachidonate release. The absence of α‐tocopherol inhibition of MeHg‐induced arachidonate release precludes a causal role for lipoperoxide‐induced PLA2 activation in this system. Moreover, MeHg induced an increased susceptibility of unilamellar vesicles to exogenous PLA2 in the presence of low Ca2+ concentrations without evidence of lipid peroxidation. [3H]Arachidonate incorporation into granule neuron phospholipids was analyzed by isocratic HPLC analysis. Relatively high proportional incorporation was found in the combined phosphatidylcholine fractions and phosphatidylinositol. With MeHg, an increase in the relative specific activity of incorporation was found in the phosphatidylinositol fraction, indicating a preferential turnover in this phospholipid species in the presence of MeHg.
Increased Production of Paired Helical Filament Epitopes in a Cell Culture System Reduces the Turnover of τ
Authors: Vincent Inez , Rosado Michelle , Kim Elaine , Davies Peter
Published: July 2010

Abstract: To investigate the regulation of posttranslational modifications of τ that might be pertinent to the production of the paired helical filament (PHF) of Alzheimer's disease, we incubated human neuroblastoma cells with the protein phosphatase inhibitor okadaic acid. This treatment results in increased immunoreactivity of τ with the monoclonal antibodies Alz‐50, PHF‐1, T3P, and NP8, a reduction in Tau‐1 immunoreactivity, and an elevation in apparent molecular weight of τ. Moreover, our data demonstrate that accumulation of phosphates in τ leads to a decrease in the turnover rate of τ in the neuroblastoma cells. It is suggested that similar build‐up of hyperphosphorylated τ in the neuronal perikarya may represent an early event in PHF formation. The present system facilitates the investigation of regulatory mechanisms governing the occurrence of PHF epitopes, their effects on neuronal cell metabolism, and possible pharmacological intervention.
Site‐Directed Mutagenesis of Active Site Residues Reveals Plasticity of Human Butyrylcholinesterase in Substrate and Inhibitor Interactions
Authors: Gnatt Averell , Loewenstein Yael , Yaron Avraham , Schwarz Mikael , Soreq Hermona
Published: July 2010

Abstract: In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site‐directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso‐OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10‐fold decreased IC50 values for DFP, iso‐OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.
Antibodies to Myelin/Oligodendrocyte‐Specific Protein and Myelin/Oligodendrocyte Glycoprotein Signal Distinct Changes in the Organization of Cultured Oligodendroglial Membrane Sheets
Authors: Dyer Charissa A. , Matthieu Jean‐Marie
Published: July 2010

Abstract: Cultured murine oligodendrocytes elaborate extensive membrane sheets that, unlike multilamellar myelin in vivo, allow the study of interactions between myelin proteins and cytoskeletal elements. This article describes the events that occur due to the interaction of specific antibodies with their respective antigens, myelin/oligodendrocyte‐specific protein (MOSP) and myelin/oligodendrocyte glycoprotein (MOG), which are expressed uniquely by oligodendrocytes. After antibody binding, surface anti‐MOSP:MOSP complexes redistribute over those cytoplasmic microtubular veins that have 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase colocalized along them. In contrast, surface anti‐MOG‐MOG complexes redistribute over internal myelin basic protein domains. Long‐term anti‐MOSP IgM exposure results in an apparent increase in number as well as thickness of microtubular structures in oligodendrocyte membrane sheets, whereas long‐term anti‐MOG exposure causes depolymerization of microtubular veins in membrane sheets. These data suggest that antibody binding to these two surface proteins elicits signals that have opposite effects on the cytoskeleton in oligodendroglial membrane sheets. Thus, it is possible that signals transduced via antibody binding may contribute to the pathogenesis of diseases affecting CNS myelin.
Nitric Oxide Synthase Expression in Glial Cells: Suppression by Tyrosine Kinase Inhibitors
Authors: Feinstein Douglas L. , Galea Elena , Cermak Jennifer , Chugh Punita , Lyandvert Liubov , Reis Donald J.
Published: July 2010

Abstract: Rat brain glial cells have the capacity to express a calcium‐independent form of nitric oxide synthase (iNOS). To test if iNOS induction required tyrosine kinase activity, we made use of genistein, a selective inhibitor of tyrosine kinases. In both primary astrocyte cultures and C6 glioma cells, the presence of genistein prevented both lipopolysaccharide‐ and cytokine‐induced NOS activity in a dose‐dependent manner. The presence of tyrphostin‐25 (10 µM), which is highly specific for tyrosine kinases, also blocked iNOS induction. Additional characterization showed that genistein blocked iNOS induction in a dose‐dependent manner (IC50 of ∼ 40 µM), that the continuous presence of genistein was not necessary to observe inhibition, and that preincubation with genistein led to higher levels of inhibition than the simultaneous addition of genistein and inducers. The decrease in iNOS activity due to genistein was accompanied by a decrease in iNOS mRNA level as detected by a specific PCR assay. These results indicate that induction of astroglial iNOS expression requires tyrosine kinase activity.
The Stability of Endogenous Tyrosine Hydroxylase Protein in PC‐12 Cells Differs from That Expressed in Mouse Fibroblasts by Gene Transfer
Authors: Wu Doris K. , Cepko Constance L.
Published: July 2010

Abstract:Previous studies have used recombinant retroviruses encoding the tyrosine hydroxylase (TH) gene to transduce various cell lines, including fibroblasts (NIH‐3T3), a pituitary tumor cell line (AtT20), and a pancreatic endocrine line (RIN). These genetically modified cells, synthesizing either 3,4‐dihydroxyphenylalanine, dopamine, or both, are potential donors for treatment of Parkinson's disease. However, the levels of TH protein in such transduced cells have been low and heterogeneous. Using several modified versions of retrovirus vectors encoding TH, we demonstrate that protein stability is an important factor governing levels of TH in NIH‐3T3 fibroblasts. Whereas low levels of TH protein were observed in infected NIH‐3T3 cells, high levels of a TH‐βgal fusion protein were found. This difference was due to a significantly longer half‐life of the TH‐βgal fusion protein relative to TH alone. However, the TH‐βgal fusion protein was found to be enzymatically inactive. We also found that the half‐life of the endogenous TH protein in PC‐12 cells is sevenfold longer than the TH protein in transduced fibroblasts, implying that a cell‐type specific regulator or mechanism may stabilize TH in catecholaminergic cells.
Cross‐Talk of the Receptors for Bradykinin, Serotonin, and ATP Shown by Single Cell Ca2+Responses Indicating Different Modes of Ca2+Activation in a Neuroblastoma × Glioma Hybrid Cell Line
Authors: Reetz Guido , Reiser Georg
Published: July 2010

Abstract:Modes of Ca2+activation by bradykinin, serotonin, and ATP and the possible receptor cross‐talk were investigated in mouse neuroblastoma × rat glioma hybrid cells (108CC15) by monitoring fura‐2 fluorescence in single cells. A transient rise of cytosolic Ca2+activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca2+stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca2+‐free medium abolished the Ca2+response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, Induces the release of Ca2+from intracellular stores. Serotonin, in contrast, activates Ca2+entry from the extracellular space. To investigate whether ATP releases Ca2+from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca2+. The application of ATP before that of bradykinin reduced the size of a following bradykinin‐induced Ca2+response in Ca2+‐free medium, but not in Ca2+‐containing medium. This suggests that bradykinin may interact with the ATP‐activated mechanism by cross‐desensitization. Possibly, bradykinin receptors are coupled to additional Ca2+stores not accessible to ATP that are refilled by extracellular Ca2+. Cyclic AMP and cyclic GMP apparently do not affect the Ca2+responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.
Disruption of Potential α‐Helix in the G Loop of the Guinea: Pig 5‐Hydroxytryptamine2Receptor Does Not Prevent Receptor Coupling to Phosphoinositide Hydrolysis
Authors: Watts Stephanie W. , Cohen Marlene L. , Mooney Patrick Q. , Johnson Bryan G. , Schoepp Darryle D. , Baez Melvyn
Published: July 2010

Abstract:Heterogeneity of the 5‐hydroxytryptamine2(5‐HT2) receptor across species has been implicated in several pharmacological and physiological studies. Although 5‐HT2receptors in the rat have been linked to increases in Phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5‐HT2receptors with Pl hydrolysis, the second messenger generally linked with 5‐HT2receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5‐HT2receptors exist between rat and guinea pig. First, we isolated partial cortical 5‐HTareceptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor‐effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5‐HT2receptor were 97% homologous. However, the guinea pig 5‐HT2receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5‐HT2receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5‐HT2receptor from guinea pig. 5‐HT and the 5‐HT2receptor agonist α‐methyl‐5‐HT increased PI hydrolysis in guinea pig cortical slices whereas the 5‐HT1creceptor agonist 5‐methyltryptamine was significantly less potent. In addition, the 5‐HT2receptor antagonists LY53857, ketanserin, and spiperone blocked 5‐HT‐stimulated Pl hydrolysis. These pharmacological data suggested that activation of the 5‐HT2receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5‐HT2receptor that influence receptor‐effector coupling were different from the rat, such differences were not critical to receptor‐effector coupling because, as in the rat, guinea pig brain 5‐HT2receptors were also coupled to PI hydrolysis.
Down's Syndrome: Up‐Regulation of β‐Amyloid Protein Precursor and τ mRNAs and Their Defective Coordination
Authors: Oyama Fumitaka , Cairns Nigel J. , Shimada Hiroyuki , Oyama Rieko , Titani Koiti , Ihara Yasuo
Published: July 2010

Abstract: Almost all patients >40 years of age with Down's syndrome (DS) develop the pathology characteristic of Alzheimer's disease: abundant β‐amyloid plaques and neurofibrillary tangles. We have investigated the gene expression of β‐amyloid protein precursor (APR) and τ in DS and age‐matched control brains and found that levels of both mRNAs were significantly elevated in DS. Such up‐regulation was not observed in two other neuronal proteins. A correlation between total APP and τ mRNA levels was also found in DS brain but distinct from the pattern observed in normal brain. Although a proportionality existed between APP‐695 mRNA and three‐repeat τ mRNA in DS, the proportionality between APP‐751 mRNA and four‐repeat τ mRNA, which is normally present, was not observed. Thus, DS brains are primarily characterized by the up‐regulation of τ mRNA as well as APP mRNA and disruption of the coordinate expression between APP‐751 and four‐repeat τ.
NMDA and Non‐NMDA Receptor Gene Expression Following Global Brain Ischemia in Rats: Effect of NMDA and Non‐NMDA Receptor Antagonists
Authors: Pellegrini‐Giampietro Domenico E. , Pulsinelli William A. , Zukin R. Suzanne
Published: July 2010

Abstract:Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous sludy, we have shown that expression of GIuR2, the kainate/a‐amino‐3‐hydroxy‐5‐ methyl‐4‐isoxazolepropionic acid (AMPA) receptor subunit that governs Ca′ permeability, is preferentially reduced in CA1 at a time point proceeding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzo(f)quinoxaline (NBQX), protects CAI neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK‐801 (a selective NMDA receptor anltagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDARI. In ischemic rats treated with saline, GIuR2 and (31uR3 mRNAs were markedly reduced in CAI but were unchanged in CA3 or dentate gyrus. GluRl and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK‐801 did not alter the ischemia‐induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modificatian of GIuR2 expression changes