bluuebunny/crossref_metadata_2025 huggingface dataset:
Ionic Strength Dependence of Calcium, Adenine Nucleotide, Magnesium, and Caffeine Actions on Ryanodine Receptors in Rat Brain
Authors: Padua R. A. , Nagy J. I. , Geiger J. D.
Published: July 2010

Abstract:[3H]Ryanodine binding studies of ryanodine receptors in brain membrane preparations typically require the presence of high salt concentrations in assay incubations to yield optimal levels of binding. Here, radioligand binding measurements on rat cerebral cortical tissues were conducted under high (1.0MKCI) and low (200 mMKCI) salt buffer conditions to determine the effects of ionic strength on receptor binding properties as well as on modulation of ligand binding by Ca2+, Mg2+, β,γ‐methylene‐adenosine 5′‐triphosphate (AMP‐PCP), and caffeine. In 1.0MKCI buffer, labeled titration/equilibrium analyses yielded two classes of binding sites with apparentKD(nM) and Bmax(fmol/mg of protein) values of 2.4 and 34, respectively, for the high‐affinity site and 19.9 and 157, respectively, for the low‐affinity site. Unlabeled titration/equilibrium measurements gave a single high‐affinity site with aKDvalue of 1.9 nM and a Bmaxvalue of 95 fmol/mg of protein. The apparentKDvalue derived from association and dissociation studies was 20 pM.Equilibrium binding was activated by Ca2+(KD/Ca2+= 14 nM), inhibited by Mg2+(IC60= 5.0 mM), and unaffected by AMP‐PCP or caffeine. In 200 mM KCI buffer conditions, labeled titration analyses gave only a single site with aKDvalue similar to and a Bmaxvalue 1.8‐fold greater than those obtained for the low‐affinity site in 1.0MKCI buffer. In unlabeled titration measurements, theKDvalue was fivefold lower, whereas the Bmaxvalue was unaffected. TheKDvalue derived from association and dissociation analysis was 2.4‐fold greater in 200 mM KCI compared with 1.0MKCI buffer conditions. In 200 mM compared with 1.0 M KCI, the potency with which Mg2+inhibited binding was increased by 3.8‐fold, whereas the affinity of the activation site for Ca2+was reduced by 13‐fold. Addition of caffeine in the presence of low salt increased the affinity of Ca2+activation by 1.7‐fold. The inhibitory effect of Mg2+on [3H]‐ryanodine binding in the presence of 200 mMKCI was reversed by AMP‐PCP and caffeine with apparent EC50values of 0.25 and 7.6 mM, respectively. Taken together, these results indicate that ionic strength is an important consideration in binding studies of brain ryanodine receptors and their interactions with modulatory agents.
Expression of Serotonin Transporter Messenger RNA in the Human Brain
Authors: Austin Mark C. , Bradley Christopher C. , Mann J. John , Blakely Randy D.
Published: July 2010

Abstract:The characterization and cellular localization of human brain mRNA encoding the serotonin transporter were investigated using northern blot analysis and in situ hybridization histochemistry. In contrast to results from rodent studies, in which single hybridizing mRNAs are detected in brain and periphery, northern analysis of human midbrain raphe tissue revealed the presence of three mRNA species absent from samples prepared from sub‐stantia nigra/ventral tegmental area. In situ hybridization studies revealed dense hybridization signal corresponding to serotonin transporter mRNA highly localized to neurons of the dorsal and median raphe nuclei and the caudal linear nucleus. No hybridization signal was observed in neurons of the substantia nigra or locus ceruleus. These findings demonstrate the first anatomical visualization of human brain serotonin transporter gene expression and reveal heterogeneity associated with serotonin transporter transcripts, similar to, but distinct from the pattern of expression visualized in human peripheral tissues.
The 140‐kDa Protein of Blood‐Brain Barrier‐Associated Pericytes Is Identical to Aminopeptidase N
Authors: Kunz Jörg , Krause Dorothee , Kremer Marian , Dermietzel Rolf
Published: July 2010

Abstract:Although ample evidence has been accumulated on the structure and functional role of endothelial cells in blood‐brain barrier mechanisms, little is known about the contribution that cerebral pericytes provide to this phenomenon. We have reported recently on a monoclonal antibody specific for cerebral pericytes at blood‐brain barrier sites. To confirm the pericytic localization of this antigen, and in order to elucidate its biochemical identity, we have performed immunocytochemical, biochemical, and molecular biological studies. By immunocytochemistry on the light microscopic as well as electron microscopic level, we provide definite evidence that the 140‐kDa antigen recognized by this monoclonal antibody is confined to cerebral pericytes, whereas endothelial cells are devoid of this antigen. N‐Terminal sequencing of the corresponding immunocrossreacting renal protein revealed that the protein detected by the monoclonal antibody is identical to aminopeptidase N. By means of the reverse transcriptase‐polymerase chain reaction, the identity of the 140‐kDa antigen as aminopeptidase N could also be verified for cerebral microvascular cells. Cerebral pericytic aminopeptidase N may be involved in neurotransmitter (enkephalin) metabolism at the blood‐brain interface. By taking into account that brain pericytes have been found to express further plasma membrane‐bound enzymes, these results strongly suggest the contribution of cerebral pericytes in the metabolic concert of the homeo‐static balance regulated by the blood‐brain barrier.
Phosphorylated Forms of Connexin43 Predominate in Rat Brain: Demonstration by Rapid Inactivation of Brain Metabolism
Authors: Hossain M. Z. , Murphy L. J. , Hertzberg E. L. , Nagy J. I.
Published: July 2010

Abstract: The gap junction protein connexin43 (Cx43) has been reported to exist as several phosphorylated forms migrating at ˜43 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis as well as an unphosphorylated 41‐kDa form. In brain, Cx43 is expressed predominantly in astrocytes and is also expressed in several other cell types. Whereas the phosphorylated forms of Cx43 predominate in heart, several studies have indicated that high levels of the unphosphorylated form of Cx43 are present in brain. Various experiments in this report indicate that the 41‐kDa molecular form in brain is a postmortem dephosphorylation product of phosphorylated Cx43. In rats killed by cranial high‐energy microwave irradiation leading to rapid inactivation of brain metabolism, Cx43 in cerebral cortex was present almost exclusively as the 43‐kDa phosphorylated form. Rapid dissection of brain followed by heat treatment or inclusion of phosphatase inhibitors during tissue homogenization also largely prevented the conversion of the 43‐to the 41‐kDa form. The 41‐kDa species was generated after alkaline phosphatase digestion of the 43‐kDa material obtained by immunoprecipitation from microwave‐irradiated brain. Immunolabeling patterns and relative regional levels of Cx43 as seen by immunohistochemical and western blot detection were the same whether or not metabolism to the 41‐kDa species was prevented. In developing rat brain, Cx43 levels in frontal cortex and brainstem increased with age, but the degree of dephosphorylation of the 43‐to the 41‐kDa form was greater at earlier ages in the brainstem. It appears that brain contains a phosphatase that may be involved in modulating the phosphorylation state of Cx43 and thus may regulate intercellular communication via astrocytic gap junctions.
Tryptophan Hydroxylase Expression Is Regulated by a Circadian Clock in Xenopus laevis Retina
Authors: Green Carla B. , Besharse Joseph C.
Published: July 2010

Abstract: A circadian clock has been localized to the photoreceptor layer in the Xenopus laevis retina. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate‐limiting enzyme in melatonin synthesis in Xenopus laevis retina is tryptophan hydroxylase. A cDNA clone coding for Xenopus tryptophan hydroxylase was isolated, characterized, and used as a probe for analysis of tryptophan hydroxylase mRNA expression. Northern blot analyses of total retinal RNA show that the tryptophan hydroxylase message levels are low in the day and higher at night. The expression of tryptophan hydroxylase mRNA is under circadian control because rhythmic changes are also seen in constant darkness, with elevated levels during the subjective night. Nuclear run‐on analysis during the first subjective day in constant darkness revealed that transcription initiation is low early in the day and increases throughout the day. Our observations suggest that the circadian clock modulates tryptophan hydroxylase gene expression. An understanding of how the circadian clock controls tryptophan hydroxylase expression may lead to a clearer understanding of the melatonin biosynthetic pathway, and possibly the clock itself.
Photoneural Regulation of Rat Pineal Nitric Oxide Synthase
Authors: Schaad Nicolas C. , Vanecek Jiri , Schulz Pierre E.
Published: July 2010

Abstract:We report here a photoneural regulation of nitric oxide synthase (NOS) activity in the rat pineal gland. In the absence of the adrenergic stimulation following constant light exposure (LL) or denervation, pineal NOS activity is markedly reduced. A maximal drop is measured after 8 days in LL. When rats are housed back in normal light‐dark (LD) conditions (12:12), pineal NOS activity returns to normal after 4 days. A partial decrease in pineal NOS activity is also observed when rats are placed for 8 days in LD 18:6 or shorter dark phases, indicating that pineal NOS activity reflects the length of the dark phase. Because it is known that norepinephrine (NE) is released at night from the nerve endings in the pineal gland and this release is blocked by exposure to light, our data suggest that NOS is controlled by adrenergic mechanisms. Our observation may also explain the lack of cyclic GMP response to NE observed in animals housed in constant light.
Glia of the Cholinergic Electromotor Nucleus of Torpedo Are the Source of the cDNA Encoding a GAT‐1‐Like GABA Transporter
Authors: Swanson Geoffrey T. , Umbach Joy A. , Gundersen Cameron B.
Published: July 2010

Abstract: A PCR‐based strategy was used to clone DNAs encoding Na+‐ and Cl−‐dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter‐1 (GAT‐1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT‐1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT‐1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.
Carbohydrate‐Binding Proteins in the Leech: II. Lactose‐Binding Protein LL35 Is Located to Neuronal and Muscle Subsets and All Epithelial Cells
Authors: Cole Robert N. , Zipser Birgit
Published: July 2010

Abstract: Leech lectin 35 (LL35) is a calcium‐independent galactoside‐binding protein with a molecular mass of 35 kDa and binding properties similar to those of calcium‐independent, galactose‐specific lectins found in vertebrates, sponges, and nematodes. LL35 was initially isolated from membranes of the leech CNS; however, large amounts of this lectin were also extracted from the rest of the leech. Using affinity‐purified antibodies to LL35, we report the immunocytochemical localization of LL35 in adult and embryonic leech. LL35 is developmentally regulated in epithelial, neuronal, and muscle tissue but is absent from glia. During embryogenesis, LL35 is highly expressed by a subset of sensory neurons, weakly expressed in epithelial cells, and absent from muscle. In the adult, LL35 is still present on the same sensory neurons but has become more abundant in epithelial cells lining the CNS and peripheral organs. LL35 also appeared on a muscle cell specifically located in the CNS but remained absent from peripheral muscle. The developmentally regulated distribution of LL35 in epithelial cells, neurons, and CNS muscles suggests a multifunctional role for this lectin with respect to these different cell types.
Neurosteroidogenesis in Rat Retinas
Authors: Guarneri P. , Guarneri R. , Cascio C. , Pavasant P. , Piccoli F. , Papadopoulos V.
Published: July 2010

Abstract: Neurosteroids (steroids synthesized in the CNS) function by modulating neurotransmission. To establish an experimental model for investigation of neurosteroid synthesis and regulation, independent of blood‐borne steroids, we examined the steroidogenic activity of isolated rat retinas. We identified progesterone, pregnenolone, dehydroepiandrosterone, desoxycorticosterone, 3α,5α‐tetrahydrodesoxycorticosterone, 3α‐hydroxy‐5α‐dihydroprogesterone, 17‐hydroxyprogesterone, and 17‐hydroxypregnenolone together with their esterified forms. As pregnenolone is the precursor of all steroids, its formation was studied in detail as an index of a steroid‐synthesizing tissue. Pregnenolone was identified further by gas chromatography coupled to mass spectrometry, and its in vitro synthesis was inhibited by lovastatin, an inhibitor of mevalonolactone and cholesterol biosynthesis. We then examined pregnenolone synthesis in the presence of mevalonolactone as a precursor of sterol formation together with lovastatin, which reduces endogenous mevalonolactone synthesis, as well as with inhibitors of pregnenolone metabolism. The incorporation of mevalonolactone into pregnenolone and its sulfate ester was time‐ and concentration‐dependent and blocked by aminoglutethimide, a competitive inhibitor of cytochrome P450 side‐chain cleavage (P450scc) enzyme. Immunocytochemical studies with a specific antibody to P450scc revealed a primary localization of the enzyme at the retinal ganglion cell layer. A less pronounced immunostaining was also seen at cells of the inner nuclear layer. Compounds known to stimulate cyclic AMP content also stimulated pregnenolone formation by rat retinas. These results demonstrate that rat retinas synthesize steroids and, for the first time, they reveal the steroidogenic ability of neuronal cells. We propose rat retinas as an in vitro model system to study neurosteroidogenesis in the CNS.
l‐3,4‐Dihydroxyphenylalanine‐Induced Dopamine Release in the Striatum of Intact and 6‐Hydroxydopamine‐Treated Rats: Differential Effects of Monoamine Oxidase A and B Inhibitors
Authors: Wachtel Stephen R. , Abercrombie Elizabeth D.
Published: July 2010

Abstract: Administration of l‐DOPA (50 mg/kg) elicits a significant increase in extracellular dopamine in striata of rats treated with the catecholaminergic neurotoxin 6‐hydroxydopamine but not in striata of intact rats. To assess the role of dopaminergic nerve terminals in determining the effects of exogenous l‐DOPA on extracellular dopamine levels in striatum, we examined the relative contributions of monoamine oxidase A and monoamine oxidase B to the catabolism of dopamine synthesized from exogenous l‐DOPA. Extracellular concentrations of dopamine and its catabolite, 3,4‐dihydroxyphenylacetic acid, were monitored with in vivo dialysis in striata of intact rats and of rats with unilateral 6‐hydroxydopamine lesions of striatal dopamine. Clorgyline (2 mg/kg), an inhibitor of monoamine oxidase A, significantly increased dopamine and decreased 3,4‐dihydroxyphenylacetic acid in intact but not in dopamine‐depleted striata. Inhibition of monoamine oxidase B with either l‐deprenyl (1 mg/kg) or Ro 19‐6327 (1 mg/kg) did not significantly affect dopamine or 3,4‐dihydroxyphenylacetic acid in striata of intact or dopamine‐depleted rats. In intact rats, administration of clorgyline in conjunction with l‐DOPA produced a >20‐fold increase in dopamine and prevented the l‐DOPA‐induced increase in 3,4‐dihydroxyphenylacetic acid. Although both l‐deprenyl and Ro 19‐6327 administered in combination with l‐DOPA elicited a small but significant increase in dopamine, levels of 3,4‐dihydroxyphenylacetic acid were not affected. In rats pretreated with 6‐hydroxydopamine, clorgyline had no significant effect on the increases in dopamine and 3,4‐dihydroxyphenylacetic acid elicited by l‐DOPA. Furthermore, neither l‐deprenyl nor Ro 19‐6327 affected l‐DOPA‐induced increases in dopamine and 3,4‐dihydroxyphenylacetic acid in dopamine‐depleted striata. The present findings indicate that deamination by monoamine oxidase A is the primary mechanism for catabolism of striatal dopamine, both under basal conditions and after administration of exogenous l‐DOPA. Loss of dopaminergic terminals eliminates this action of monoamine oxidase A but does not enhance deamination by monoamine oxidase B. These data support a model in which exogenous l‐DOPA elicits enhanced extracellular accumulation of dopamine in the dopamine‐depleted striatum because some transmitter synthesis occurs at nondopaminergic sites and the dopamine terminals that normally take up and catabolize this pool of transmitter are absent.
Characterization, Distribution, and Protein Kinase C‐Mediated Regulation of [35S]Glutathione Binding Sites in Mouse and Human Spinal Cord
Authors: Lanius Ruth A. , Shaw Christopher A. , Wagey Ravenska , Krieger Charles
Published: July 2010

Abstract: We have characterized a high‐affinity [35S]‐glutathione ([35S]GSH) binding site in mouse and human spinal cord. [35S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High‐affinity [35S]GSH binding was saturable, showing a Bmax of 72 fmol/mg of protein and a KD of 3.0 nM for mouse spinal cord and a Bmax of 52 fmol/mg of protein and a KD of 1.6 nM for human spinal cord. [35S]GSH binding was displaceable by GSH, l‐cysteine, and S‐hexyl‐GSH, but not by glutamate, glycine, or NMDA. These [35S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [35S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13‐diacetate for 1 h before ligand binding. Phorbol ester treatment increased [35S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine, a general protein kinase inhibitor. [35S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.
Evidence that Extracellular Concentrations of Dopamine Are Regulated by Noradrenergic Neurons in the Frontal Cortex of Rats
Authors: Pozzi L. , Invernizzi R. , Cervo L. , Vallebuona F. , Samanin R.
Published: July 2010

Abstract: Experiments were performed to confirm that noradrenergic terminals regulate extracellular concentrations of dopamine (DA) in the frontal cortex of rats. The effects of 20 mg/kg 1‐[2‐[bis(4‐fluorphenyl)methoxy]‐ethyl]‐4‐(3‐phenylpropyl)piperazine (GBR 12909), a selective inhibitor of DA uptake, and 2.5 mg/kg desipramine (DMI) on the extracellular concentrations of DA in the frontal cortex and striatum were studied in rats given 6‐hydroxydopamine (6 µg/µl) bilaterally into the locus coeruleus to destroy noradrenergic terminals. GBR 12909 increased dialysate DA similarly in the striatum of vehicle and 6‐hydroxydopamine‐treated rats, whereas in the frontal cortex it raised DA concentrations only in lesioned animals. DMI raised extracellular DA concentrations in the frontal cortex but not in the striatum of controls. The effect of DMI on cortical DA was abolished by the 6‐hydroxydopamine lesion. GBR 12909, at a subcutaneous dose of 20 mg/kg, further increased cortical dialysate DA in rats given DMI intraperitoneally at 20 mg/kg or through the probe at 10−5 mol/L. The data support the hypothesis of an important regulation of the extracellular concentrations of DA in the frontal cortex by noradrenergic terminals.
Characterization of Basal and Morphine‐Induced Histamine Release in the Rat Periaqueductal Gray
Authors: Barke Kim E. , Hough Lindsay B.
Published: July 2010

Abstract: Previous studies have shown that antinociceptive doses of systemic morphine increase extracellular histamine (HA) levels in the rat periaqueductal gray (PAG), although the cellular origin of basal and morphine‐induced HA release in the PAG is unknown. Treatment with α‐fluoromethylhistidine (FMH; 100 mg/kg, i.p.), the irreversible inhibitor of histidine decarboxylase, decreased basal HA release by a maximum of 80% and prevented morphine‐induced HA release in the PAG. In addition, perfusion of this area with the sodium channel blocker tetrodotoxin (10−6M) decreased basal HA release by a maximum of 57% from baseline levels. When the perfusion medium was modified by substitution of magnesium for calcium, extracellular HA levels in the PAG decreased by a maximum of 72%, and morphine‐induced HA release was prevented. Thioperamide (5 mg/kg, i.p.), an H3 antagonist, increased HA release in the PAG to a maximum of 249% within the first 30–60‐min period. Taken together, these results suggest that basal and morphine‐induced HA release in the rat PAG have a neuronal origin.
Autoradiographic Demonstration of Increased α2‐Adrenoceptor Agonist Binding Sites in the Hippocampus and Frontal Cortex of Depressed Suicide Victims
Authors: González Antonio M. , Pascual Julio , Meana J. Javier , Barturen Fernando , Del Arco Carmen , Pazos Angel , García‐Sevilla Jesús A.
Published: July 2010

Abstract: To examine directly in the brain the status of α2‐adrenoceptors in major depression, the specific binding of the agonist [3H]UK 14304 was measured by quantitative receptor autoradiography in the hippocampus and frontal cortex of suicide victims (n = 17) with a retrospective diagnosis of depression (n = 7) or other psychiatric disorders (n = 10) as well as of matched control subjects (n = 9). In suicide victims, a significant increase in the number of α2‐adrenoceptors was found in the CA1 field (40%) and dentate gyrus (20%) of the hippocampus and in the external layers I (33%) and II (31%) of the frontal cortex, compared with that in matched controls. In depressed suicide victims, the increase in α2‐adrenoceptors in the CA1 field (57%) was significantly greater (24%, p < 0.05) than that observed in the group of suicide victims with other diagnoses (26%). In the same depressed suicide victims, the increase in cortical α2‐adrenoceptors was restricted to layer I (34%) and it was equivalent to that found in layer I (33%) of suicide victims with other diagnoses. The results indicate that suicide is associated with increases in the high‐affinity state of brain α2‐adrenoceptors and that there is a pronounced localized increase of this inhibitory receptor in the hippocampus of depressed suicide victims.
Induction and Regulation of Nitric Oxide Synthase in Retinal Müller Glial Cells
Authors: Goureau Olivier , Hicks David , Courtois Yves , De Kozak Yvonne
Published: July 2010

Abstract: Müller glial cells from the rat retina were examined for their capacity to produce nitric oxide (NO). Treatment of retinal Müller glial (RMG) cells with lipopolysaccharide (LPS), interferon‐γ, and tumor necrosis factor‐α induced NO synthesis as determined by nitrite release in media. Simultaneous addition of LPS, interferon‐γ, and tumor necrosis factor‐α caused the largest increase in NO synthesis. NO biosynthesis was detected after 12 h and was dependent on the dose of LPS, interferon‐γ, and tumor necrosis factor‐α. Stereoselective inhibitors of NO synthase (NOS), cycloheximide and transforming growth factor‐β, blocked cytokine‐induced NO production. Cytosol from LPS/cytokine‐treated RMG cultures, but not from unstimulated cultures, produced a calcium/calmodulin‐independent conversion of l‐arginine to l‐citrulline that was completely blocked by NOS inhibitor. The expression of NOS in RMG cells was confirmed by northern blot analysis, in which stimulation of these cells led to an increase in NOS mRNA levels. We conclude that RMG cells can express an inducible form of NOS similar to the macrophage isoform. High NO release from activated RMG cells might represent a protection from infection but may also contribute to the development of retinal pathologies.
Antipeptide Polyclonal Antibodies that Recognize a Substance P‐Binding Site in Mammalian Tissues: A Biochemical and Immunocytochemical Study
Authors: Bret‐Dibat J. L. , Zouaoui D. , Déry O. , Zerari F. , Grassi J. , Maillet S. , Conrath M. , Couraud J. Y.
Published: July 2010

Abstract: We used complementary peptide methodology to obtain antibodies against the receptor for the neuropeptide substance P, specifically directed at the ligand‐binding domain. Rabbits were immunized with two distinct peptides derived from the sequence of the RNA complementary to the mRNA for substance P. Binding experiments revealed that antipeptide polyclonal antibodies were able to recognize, through their paratope, a specific binding site on the rat parotid cell membranes. Substance P and antibodies competed for this binding site, because preincubation of membranes in the presence of substance P significantly reduced antibody binding, and conversely, preincubation of membranes in the presence of antibodies partly inhibited the binding of radioiodinated substance P. Immunocytochemical experiments performed on the rat cervical spinal cord show that the distribution of labeling by antibodies is similar to that observed by conventional autoradiography using 125I‐substance P. Here again, control experiments demonstrated that antibodies and substance P were competing for the same binding site on the spinal cord. These biochemical and immunocytochemical data indicate that antipeptide antibodies recognize a substance P membrane binding site in nervous and nonnervous mammalian tissues. This site is likely to correspond to the NK1 specific receptor for substance P.
Vasoactive Intestinal Peptide and Forskolin Stimulate Interleukin 6 Production by Rat Cortical Astrocytes in Culture via a Cyclic AMP‐Dependent, Prostaglandin‐Independent Mechanism
Authors: Grimaldi Maurizio , Pozzoli Giacomo , Navarra Pierluigi , Preziosi Paolo , Schettini Gennaro
Published: July 2010

Abstract: In this study we analyzed the involvement of the cyclic AMP (cAMP)‐protein kinase A system in the regulation of interleukin 6 production by cultured cortical astrocytes. Vasoactive intestinal peptide strongly increased, in a dose‐dependent manner, interleukin 6 production. This effect was reduced when protein kinase A was blocked by KT‐5720; it was not affected by calphostin C, a protein kinase C inhibitor. Forskolin caused a concentration‐dependent increase in interleukin 6 release that was also inhibited by KT‐5720. Because prostaglandins are believed to play a role in interleukin 6 production, we tried to determine whether the stimulatory effects of vasoactive intestinal peptide and forskolin on cytokine release might be mediated by stimulation of prostaglandin production in cortical astrocytes. Vasoactive intestinal peptide did not increase the production of either prostaglandin E2 or F2α. Conversely, forskolin concentration‐dependently stimulated the production of both prostaglandins, an effect that was blocked by indomethacin. Indomethacin did not affect either vasoactive intestinal peptide‐ or forskolin‐stimulated interleukin 6 production. To exclude the possibility that prostaglandins participate in interleukin 6 production induced by forskolin, we tested prostaglandins E2 and F2α. The former was completely ineffective in eliciting the cytokine production, whereas prostaglandin F2α slightly increased interleukin 6 production only at the highest concentrations. 8‐Bromo‐cAMP and dibutyryl‐cAMP stimulated interleukin 6 production to a lesser extent than vasoactive intestinal peptide and forskolin. In conclusion, we provide evidence that vasoactive intestinal peptide increases interleukin 6 production by astrocytes through the stimulation of the cAMP‐protein kinase A pathway, an effect that is reproduced by cAMP analogues. In addition, we point out that prostaglandins are not involved in vasoactive intestinal peptide‐ and forskolin‐mediated induction of interleukin 6 production in cultured astrocytes.
Secreted Form of Amyloid β/A4 Protein Precursor (APP) Binds to Two Distinct APP Binding Sites on Rat B103 Neuron‐Like Cells Through Two Different Domains, but Only One Site Is Involved in Neuritotropic Activity
Authors: Ninomiya H. , Roch J.‐M. , Jin L.‐W. , Saitoh T.
Published: July 2010

Abstract: Recent studies have identified the Alzheimer's disease amyloid β/A4 protein precursor (APP) as a trophic and/or tropic protein on several types of cells, including fibroblasts, primary culture neurons, PC12 cells, and B103 neuron‐like cells. Many trophic proteins bind heparin, and it is believed that the heparin‐binding domain is crucial for the trophic activity of these proteins. APP also binds heparin. The current studies were undertaken to examine the hypothesis that the neuritotropic activity of APP requires heparin binding. It was found that APP produced in E. coli bound B103 cells through detergent‐extractable molecules. Approximately 50% of the binding sites were heparinase‐sensitive, and heparin and heparan sulfate competed for APP binding to these sites. The heparinase‐insensitive sites were recognized by a stretch of 17 amino acids of APP (residues 319–335) that contains the neuritotropic activity of APP. A mutant APP with a deletion at this site was capable of binding to the heparinase‐sensitive sites, although this molecule was not neuritotropic to B103 neuron‐like cells. Therefore, the neuritotropic site and the heparin‐binding site are distinct in APP, and the neuritotropic effect of APP is produced through its binding to detergent‐extractable and heparinase‐insensitive sites.
Increased Extracellular Serotonin in Rat Brain After Systemic or Intraraphe Administration of Morphine
Authors: Tao Rui , Auerbach Sidney B.
Published: July 2010

Abstract: The effect of morphine on serotonin (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) in the CNS of unanesthetized rats was investigated by microdialysis. Morphine was administered either subcutaneously, by local perfusion into the diencephalon, or by intraraphe microinjection. Systemic administration of morphine resulted in a significant increase in both extracellular 5‐HT and 5‐HIAA in the diencephalon. The effect of morphine on 5‐HT was dose dependent during local perfusion of the diencephalon with inhibitors of uptake or monoamine oxidase. Systemic morphine also produced significant increases in extracellular 5‐HT in the striatum and hippocampus during uptake inhibition. The site of opioid effects on 5‐HT was tested by locally perfusing morphine into the diencephalon. This had no effect on 5‐HT or 5‐HIAA. In contrast, intraraphe injection of morphine caused a dose‐dependent increase in extracellular 5‐HT and 5‐HIAA in the diencephalon. These results suggest that systemic morphine induces an increase in 5‐HT release in widespread areas of the forebrain. This appears to be due to an effect on 5‐HT cell bodies and not on 5‐HT nerve endings in projection sites.
Physiological Elevations of Glucocorticoids Potentiate Glutamate Accumulation in the Hippocampus
Authors: Stein‐Behrens Becky A. , Lin Walter J. , Sapolsky Robert M.
Published: July 2010

Abstract: Glucocorticoids (GCs) are secreted during stress and can damage the hippocampus over the course of aging and impair the capacity of hippocampal neurons to survive excitotoxic insults. Using microdialysis, we have previously observed that GCs augment the extracellular accumulation of glutamate and aspartate in the hippocampus following kainic acid‐induced seizures. In that study, adrenalectomized rats maintained on minimal GC concentrations were compared with those exposed to GCs elevated to near‐pharmacological levels. We wished to gain insight into the physiological relevance of these observations. Thus, we have examined the effects of GCs over the normal physiological range on glutamate and aspartate profiles; this was done by implanting adrenalectomized rats with GC‐secreting pellets, which produce stable and controllable circulating GC concentrations. We observe that incremental increases in GC concentrations cause incremental increases in glutamate accumulation before the kainic acid insult, as well as in the magnitude of the glutamate response to kainic acid. Elevating GC concentrations from the circadian trough to peak doubled cumulative glutamate accumulation, whereas a rise into the stress range caused a fourfold increase in accumulation. Similar, although smaller, effects also occurred with aspartate accumulation (as well as with taurine but not glutamine accumulation). These data show that the highly elevated GC concentrations that accompany neurological insults such as seizure or hypoxia‐ischemia will greatly exacerbate the glutamate accumulation at that time. Furthermore, stress levels of GCs augmented glutamate accumulation even in the absence of an excitotoxic insult, perhaps explaining how sustained stress itself damages the hippocampus. Finally, even the moderately ėlevated basal GC concentrations that typically occur in aged rats augmented glutamate accumulation, perhaps explaining how GCs damage the hippocampus over the course of normal aging.
Ubiquinone‐10 Protects Neurons from Virus‐Induced Degeneration
Authors: Edlund Conny , Holmberg Kristina , Dallner Gustav , Norrby Erling , Kristensson Krister
Published: July 2010

Abstract: Cultured neurons from rat dorsal root ganglia and cerebral cortex were infected with Sendai virus, which gives a productive replication with lysis of most neurons, and with the RW strain of mumps virus, which undergoes defective replication causing degeneration of only 30–40% of the neurons within 5 days after initial infection. In Sendai virus‐infected cells the amount of polyisoprenoid lipids was enhanced. In mumps virus‐infected cultures there were transient reductions in the contents of cholesterol, dolichol, and ubiquinone‐9 in the cultures, whereas the reduction in the ubiquinone‐10 level was progressive, reaching 20% of its original value 21 days after infection. Treatment of mumps virus‐infected cultures with ubiquinone‐10 protected the neurons from degeneration, whereas no effects were observed on exposure to ubiquinone‐9. Linolenic acid (18:3) and arachidonic acid (20:4), but not myristic acid (14:0) and palmitic acid (16:0), also had significant neuroprotective effects.
Dopamine‐Releasing Action of 6R‐l‐erythro‐Tetrahydrobiopterin: Analysis of Its Action Site Using Sepiapterin
Authors: Koshimura Kunio , Miwa Soichi , Watanabe Yasuyoshi
Published: July 2010

Abstract: Recently, we reported that 6R‐l‐erythro‐tetrahydrobiopterin (6R‐BH4), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine‐releasing action independent of its cofactor activity. Here we attempted to determine whether 6R‐BH4 acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, an immediate precursor of 6R‐BH4 in the salvage pathway, was used to selectively increase the intracellular 6R‐BH4 levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6R‐BH4) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4‐dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor α‐methyl‐p‐tyrosine. Administration of 6R‐BH4 increased extracellular levels of reduced biopterin, DOPA, and DA. After pretreatment with α‐methyl‐p‐tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6R‐BH4 stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.
Membrane Topology of the GluR1 Glutamate Receptor Subunit: Epitope Mapping by Site‐Directed Antipeptide Antibodies
Authors: Molnár Elek , McIlhinney R. A. Jeffrey , Baude Agnès , Nusser Zoltán , Somogyi Péter
Published: July 2010

Abstract: In order to define the membrane topology of the GluR1 glutamate receptor subunit, we have examined the location of epitopes. Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluR1 glutamate receptor subunit. Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N‐terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C‐terminal part are found at the intracellular face of the postsynaptic membrane. Furthermore, antibodies to the N‐terminal residues 253–267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C‐terminal residues 877–889 increased about 1.6‐fold following permeabilization. Our data suggest that the N‐ and C‐terminal regions are located on the opposite side of the membrane and, therefore, the GluR1 subunit probably has an odd number of membrane spanning segments. The antibody cross‐reactivities in different species and their effect on ligand binding activity were also established.
3,5‐Dihydroxyphenylglycine Is a Highly Selective Agonist for Phosphoinositide‐Linked Metabotropic Glutamate Receptors in the Rat Hippocampus
Authors: Schoepp Darryle D. , Goldsworthy John , Johnson Bryan G. , Salhoff Craig R. , Baker S. Richard
Published: July 2010

Abstract: Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein‐coupled glutamate receptors that are linked to multiple second messenger systems in the CNS. In this study the selectivity of mGluR agonists for different mGluR second messenger effects was characterized in slices of the rat hippocampus. The mGluR agonists (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid and (2S,3S,4S)α‐(carboxycyclopropyl)glycine produced multiple effects on second messengers that included enhanced phosphoinositide hydrolysis in both adult and neonatal rat hippocampus, inhibition of forskolin‐stimulated cyclic AMP (cAMP) formation in adult tissue, and increases in basal cAMP formation in the neonatal hippocampus. In contrast, 3,5‐dihydroxyphenylglycine was potent and effective in increasing phosphoinositide hydrolysis in both adult and neonatal hippocampus but unlike the other mGluR agonists did not inhibit forskolin‐stimulated cAMP formation (in the adult) or substantially enhance basal cAMP formation (in the neonate). Thus, in the rat hippocampus mGluR agonist‐mediated increases or decreases in cAMP formation are not secondary to mGluR‐mediated changes in phosphoinositide hydrolysis. Furthermore, 3,5‐dihydroxyphenylglycine can be used to activate subpopulations of mGluRs coupled to phosphoinositide hydrolysis with minimal effects on cAMP‐mGluR second messenger systems.
Regulation of μ‐Opioid Receptor mRNA in Rat Globus Pallidus: Effects of Enkephalin Increases Induced by Short‐ and Long‐Term Haloperidol Administration
Authors: Delfs Jill M. , Yu Lei , Ellison Gaylord D. , Reisine Terry , Chesselet Marie‐Françoise
Published: July 2010

Abstract: The mRNA encoding μ‐opioid receptors is expressed in neurons of the globus pallidus, a region of the basal ganglia that receives a dense enkephalinergic innervation from the striatum. The regulation of the mRNAs encoding the opioid peptide enkephalin in the striatum and the μ‐opioid receptor in the globus pallidus was examined with in situ hybridization histochemistry following short‐ or long‐term haloperidol treatments, which alter striatal enkephalin mRNA levels. Animals were administered haloperidol daily for 3 or 7 days (1 mg/kg, s.c.) or continuously for 8 months (1 mg/kg, depot followed by oral). Enkephalin and μ‐opioid receptor mRNA levels were unchanged after 3 days of haloperidol treatment. In contrast, the enkephalin mRNA level was increased in the striatum, and μ‐opioid receptor mRNA levels were markedly decreased in the globus pallidus after 7 days of haloperidol administration. Similar effects were observed in rats treated with haloperidol for 8 months. The results provide the first evidence of regulation of μ‐opioid receptor mRNA in vivo.
m1–m5 Muscarinic Receptor Distribution in Rat CNS by RT‐PCR and HPLC
Authors: Wei Jian , Walton Elizabeth A. , Milici Antonio , Buccafusco Jerry J.
Published: July 2010

Abstract: Five muscarinic receptor genes (m1–m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase‐free DNase to remove any residual genomic DNA. Total RNA (1 µg) was reverse‐transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)‐amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR‐amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus‐hypothalamus and pons‐medulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT‐PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.
Cyclic AMP‐Dependent Protein Kinase Regulates Basal and Cyclic AMP‐Stimulated but Not Phorbol Ester‐Stimulated Transcription of the Tyrosine Hydroxylase Gene
Authors: Kim Kwang‐Soo , Tinti Cristina , Song Bai , Cubells Joseph F. , Joh Tong H.
Published: July 2010

Abstract: To define the precise role of cyclic AMP (cAMP)‐dependent protein kinase (PKA) in transcriptional regulation of the tyrosine hydroxylase (TH) gene, we performed transient cotransfection analyses of a reporter construct containing the upstream 2,400 bp sequence of the rat TH gene with expression plasmids encoding a heat‐stable specific inhibitor of PKA (PKI), a mutant regulatory subunit of PKA, or the catalytic subunit of PKA. Inhibition of PKA activity by expression of either PKI or mutant regulatory subunit blocked cAMP‐stimulated induction and reduced basal transcription of the TH‐reporter construct. Expression of the catalytic subunit of PKA induced the expression of the TH‐reporter construct up to 50‐fold in a dose‐dependent manner. Primer extension analysis confirmed that PKA‐mediated induction of TH‐reporter expression occurred at the correct transcription initiation site. Expression of PKI did not affect induction following phorbol ester treatment, suggesting that PKA and protein kinase C (PKC) induce TH transcription by independent mechanisms. Finally, a double mutation within the cAMP response element (CRE) of TH2400‐CAT diminished its basal and forskolin‐stimulated transcription to the level of the promoterless plasmid, pBLCAT3, but did not alter the induction following treatment with phorbol ester, indicating that the CRE is not required for PKC‐mediated transcriptional induction. Our results indicate that PKA, via the CRE, plays a crucial role for basal and cAMP‐inducible transcription of the TH gene.
Cell Size and the Heat‐Shock Response in Rat Brain
Authors: Morrison‐Bogorad M. , Pardue S. , McIntire D. D. , Miller E. K.
Published: July 2010

Abstract: The expression of mRNAs encoding two members of the heat‐shock protein 70 family, the constitutively‐expressed heat‐shock cognate (hsc70) mRNA and the strictly heat‐inducible (hsp70) mRNA, was quantitated in cerebellar and hippocampal cells of rats 3 h after amphetamine‐induced or heat‐induced hyperthermia. Intracellular heat‐shock mRNA levels in specific cell types were compared with those of total polyadenylic acid [poly(A)] mRNA or 18S rRNA in the same cell type. Levels of poly(A) mRNAs, 18S rRNAs, and hsc70 mRNAs were highest in large neurons and lowest in glia. hsp70 mRNAs were also present at highest levels in large neurons, suggesting that hsp70 mRNAs accumulated as rapidly in these cell types as they did in small neurons and glia. However, compared with levels of intracellular poly(A) mRNAs or levels of rRNAs, large neurons contained two‐ to 12‐fold lower levels of hsp70 mRNAs than neurons of intermediate size and five‐ to 30‐fold lower levels than glia. These results suggest that hsp70 mRNAs accumulated as rapidly in large neurons as in small neurons and glia, but that the large size of these neurons precluded intracellular hsp70 mRNA concentrations increasing as quickly. The susceptibility of large neurons to stress‐induced cell death could be due, in part, to their inability to synthesize rapidly hsp70 in sufficient amounts to protect these cells from the initial molecular consequences of stress.
Distribution and Ontogeny of 1S,3R‐1‐Aminocyclopentane‐1,3‐Dicarboxylic Acid‐Sensitive and Quisqualate‐Insensitive [3H]Glutamate Binding Sites in the Rat Brain
Authors: Wright Rebecca A. , McDonald John W. , Schoepp Darryle D.
Published: July 2010

Abstract: Displacement of [3H]glutamate by 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid‐sensitive [3H]glutamate binding was found in the presence of 100 mM bromide ions and in the absence of calcium ions. Under these conditions, [3H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11‐day‐old) and adult rat brain, this [3H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid‐sensitive and quisqualate‐insensitive [3H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [3H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid but insensitive to quisqualate. Expression of [3H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [3H]glutamate is labeling a distinct population of metabotropic glutamate receptors.