bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Regulation by Methylenedioxymethamphetamine of 5‐Hydroxytryptamine1A Receptor Density and mRNA Expression in Rat Hippocampus, Frontal Cortex, and Brainstem: The Role of Corticosteroids
Authors: Aguirre Norberto , Frechilla Diana , García‐Osta Ana , Lasheras Berta , Del Río Joaquín
Published: July 2010

Abstract: The present study examined the effects of repeated administration to rats of 3,4‐methylenedioxymethamphetamine (MDMA, “Ecstasy”) on 5‐hydroxytryptamine1A (5‐HT1A) receptor density and mRNA expression in the hippocampus, frontal cortex, and brainstem. As expected, 7 days after subacute MDMA administration (20 mg/kg i.p. twice daily for 4 consecutive days) 5‐HT content was markedly reduced (−70%) in the hippocampus and the frontal cortex. 5‐HT1A receptor density was increased in the frontal cortex by 23% and decreased in the hippocampus and the brainstem by 25%. These changes correlated with an enhanced or diminished 5‐HT1A receptor mRNA expression in the three regions studied. To examine the influence of corticosteroids on these changes, adrenalectomized (ADX) rats received the same dosage regimen as above. Adrenalectomy by itself did not modify 5‐HT content in the brain regions examined and increased 5‐HT1A receptor density in the hippocampus (+20%) but produced no change in the frontal cortex and brainstem. Adrenalectomy also prevented MDMA‐induced changes in receptor number in the hippocampus and brainstem but not in the frontal cortex. Dexamethasone (1 mg/kg/day i.p.) administered for 7 consecutive days reversed the effects of adrenalectomy in the hippocampus but not in the frontal cortex. In the brainstem, MDMA no longer reduced 5‐HT1A receptor number in ADX rats, but a significant reduction was restored when ADX animals received the glucocorticoid treatment. The present data show that MDMA may affect 5‐HT1A receptors in a regionally dependent manner, notably through a drug effect on corticosterone release, which attenuates 5‐HT1A receptor gene transcription selectively in the hippocampus.
Identification of Heparin‐Binding Domains in the Amyloid Precursor Protein of Alzheimer's Disease by Deletion Mutagenesis and Peptide Mapping
Authors: Clarris Heidi J. , Cappai Roberto , Heffernan Damien , Beyreuther Konrad , Masters Colin L. , Small David H.
Published: July 2010

Abstract: Recent studies have shown that the binding of the amyloid protein precursor (APP) of Alzheimer's disease to heparan sulfate proteoglycans (HSPGs) can modulate a neurite outgrowth‐promoting function associated with APP. We used three different approaches to identify heparin‐binding domains in APP. First, as heparin‐binding domains are likely to be within highly folded regions of proteins, we analyzed the secondary structure of APP using several predictive algorithms. This analysis showed that two regions of APP695 contain a high degree of secondary structure, and clusters of basic residues, considered mandatory for heparin binding, were found principally within these regions. To determine which domains of APP bind heparin, deletion mutants of APP695 were prepared and analyzed for binding to a heparin affinity column. The results suggested that there must be at least two distinct heparin‐binding regions in APP. To identify novel heparin‐binding regions, peptides homologous to candidate heparin‐binding domains were analyzed for their ability to bind heparin. These experiments suggested that APP contains at least four heparin‐binding domains. The presence of more than one heparin‐binding domain on APP suggests the possibility that APP may interact with more than one type of glycosaminoglycan.
Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures
Authors: LeBlanc Andréa C. , Papadopoulos Maria , Bélair Caroline , Chu William , Crosato Milena , Powell Jaqueline , Goodyer Cynthia G.
Published: July 2010

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C‐terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher‐molecular‐weight fragments are more abundant. The level of amyloidogenic 4‐kDa Aβ exceeds that of nonamyloidogenic 3‐kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C‐terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.
Synaptic Vesicle Ultrastructural Changes in the Rat Hippocampus Induced by a Combination of α‐Linolenate Deficiency and a Learning Task
Authors: Yoshida S. , Yasuda A. , Kawazato H. , Sakai K. , Shimada T. , Takeshita M. , Yuasa S. , Kobayashi T. , Watanabe S. , Okuyama H.
Published: July 2010

Abstract: Rats fed either a safflower oil (α‐linolenate‐deficient) or a perilla oil (α‐linolenate‐sufficient) diet through two generations (F1) showed significant differences in the brightness‐discrimination learning task. In this task, correct responses were lever‐pressing responses, which were reinforced with dietary pellets, and incorrect responses were those with no reinforcement. The inferior learning performance in the safflower oil group was caused mainly by the inferior ability to rectify the incorrect responses through the learning sessions. In the safflower oil group after the learning task, the average densities of synaptic vesicles in the terminals of the hippocampus CA1 region were decreased by nearly 30% as compared with those in the perilla oil group, and it is notable that this difference was not detected without the learning task. These results suggest that dietary oil‐induced morphological changes in synapses in the hippocampus of rats are related to the differential learning performance and that the turnover rate of synaptic vesicles in the hippocampus may be an important factor affecting learning performance.
Characterization of a Sulfoglucuronyl Carbohydrate Binding Protein in the Developing Nervous System
Authors: Nair Sukumaran M. , Jungalwala Firoze B.
Published: July 2010

Abstract: The developmentally regulated and stage‐specifically expressed HNK‐1 carbohydrate found on sulfoglucuronylglycolipids (SGGLs) and certain glycoproteins has been proposed to be involved in neural cell adhesion and recognition processes through its interaction with protein “receptors.” We have isolated and purified a ∼30‐kDa SGGL‐binding protein (SBP‐1) from neonatal rat brain. SBP‐1 specifically bound to SGGLs and sulfatide both in solid‐phase immunobinding and high‐performance thin‐layer chromatography‐immunooverlay assays. N‐terminal sequence analysis showed that SBP‐1 is similar to an adhesive neurite outgrowth promoting protein amphoterin. Desulfation of SGGLs resulted in abolition of SBP‐1 binding. However, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect, suggesting requirement of the carbohydrate‐linked sulfate group for SBP‐1 binding. The binding of SBP‐1 to SGGLs was specifically inhibited by HNK‐1 antibody but not by other IgM antibodies. The binding of SBP‐1 to sulfatide, however, was not inhibited by HNK‐1 antibody. Heparin, fucoidan, and dextran sulfate (50K) also inhibited the binding of SBP‐1 to SGGLs. During development of the rat cerebral cortex, the level of SBP‐1 decreased after embryonic day 18 to an almost undetectable level by postnatal day 10; whereas in the cerebellum, the expression of SBP‐1 was maximal at postnatal day 7. SBP‐1 also bound specifically to the HNK‐1 glycoproteins isolated from rat brain by HNK‐1 immunoaffinity chromatography. Proteins without HNK‐1 carbohydrate did not bind SBP‐1. The binding to HNK‐1 glycoproteins was inhibited by HNK‐1 antibody, but not by other IgM antibodies, indicating that the binding was mediated through the HNK‐1 carbohydrate moiety of the proteins. The interaction and coexpression of SBP‐1 with SGGLs and HNK‐1 glycoproteins, during the perinatal brain development, suggest a functional role for this protein.
Inhibitory Effect of Several Nitric Oxide‐Generating Compounds on Purified Na+,K+‐ATPase Activity from Porcine Cerebral Cortex
Authors: Sato Tomoaki , Kamata Yoshiko , Irifune Masahiro , Nishikawa Takashige
Published: July 2010

Abstract: Nitric oxide (NO)‐generating compounds (NO donors) such as sodium nitroprusside, S‐nitroso‐N‐acetylpenicillamine, S‐nitroso‐l‐glutathione, 3‐morpholinosyndnonimine (SIN‐1), (dl)‐(E)‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide, and 1‐hydroxy‐2‐oxo‐3‐(N‐methyl‐3‐aminopropyl)‐3‐methyl‐1‐triazene inhibited the Na+,K+‐ATPase activity purified from porcine cerebral cortex. NO‐reducing or ‐scavenging agents, such as superoxide dismutase or N‐(dithiocarbamate)‐N‐methyl‐d‐glucamine sodium salt, l‐ascorbic acid, and sulfhydryl (SH) compounds, such as dithiothreitol or the reduced form of glutathione, but not α‐tocopherol, prevented the inhibition of the enzyme activity by all NO donors except sodium nitroprusside. Enzyme inhibition could also be reversed by these SH compounds, but not by superoxide dismutase, l‐ascorbic acid, and α‐tocopherol. 2‐(4‐Carboxyphenyl)‐4,4,5,5‐tetramethylimidazolin‐1‐oxyl 3‐oxide (PTIO), which is able to scavenge NO radicals and generate nitrogen dioxide radicals (•NO2), potentiated the inhibition of this enzyme activity induced by all NO donors (except SIN‐1). PTIO did not potentiate, but rather attenuated, the SIN‐1‐induced inhibition. SIN‐1 has been reported to release both NO and superoxide and thereby to rapidly form peroxynitrite (ONOO−). These potentiated and attenuated inhibitions of the enzyme activity induced by PTIO plus all of the NO donors except sodium nitroprusside were prevented by SH compounds, but not by superoxide dismutase, l‐ascorbic acid, and α‐tocopherol. These results suggest that NO donors may release NO or NO‐derived products, presumably •NO2 and ONOO−, and may inhibit the Na+,K+‐ATPase activity by interacting with a SH group at the active site of the enzyme.
Molecular Cloning of a Preprohormone from Hydra magnipapillata Containing Multiple Copies of Hydra‐LWamide (Leu‐Trp‐NH2) Neuropeptides: Evidence for Processing at Ser and Asn Residues
Authors: Leviev Ilia , Williamson Michael , Grimmelikhuijzen Cornelis J. P.
Published: July 2010

Abstract: The simple, freshwater polyp Hydra is often used as a model to study development in cnidarians. Recently, a neuropeptide, <Glu‐Gln‐Pro‐Gly‐Leu‐Trp‐NH2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a polyp. Here, we have cloned a preprohormone from Hydra magnipapillata containing 11 (eight different) immature neuropeptide sequences that are structurally related to the metamorphosis‐inducing neuropeptide from sea anemones. During the final phase of our cloning experiments, another research team independently isolated and sequenced five of the neuropeptides originally found on the preprohormone. Comparison of these mature neuropeptide structures with the immature neuropeptide sequences on the preprohormone shows that most immature neuropeptide sequences are preceded by Ser or Asn residues, indicating that these residues must be novel processing sites. Thus, the structure of the Hydra prepro‐hormone confirms our earlier findings that cnidarian pre‐prohormones contain unusual or novel processing sites. Nearly all neuropeptide copies located on the Hydra preprohormone will give rise to mature neuropeptides with a C‐terminal Gly‐Leu‐Trp‐NH2 sequence (the most frequent one being Gly‐Pro‐Pro‐Pro‐Gly‐Leu‐Trp‐NH2; Hydra‐LWamide I; three copies). Based on their structural similarities with the metamorphosis‐inducing neuropeptide from sea anemones, the mature peptides derived from the Hydra‐LWamide preprohormone are potential candidates for being developmentally active neurohormones in Hydra.
Buspirone Enhances Duloxetine‐ and Fluoxetine‐Induced Increases in Dialysate Levels of Dopamine and Noradrenaline, but Not Serotonin, in the Frontal Cortex of Freely Moving Rats
Authors: Gobert A. , Rivet J.‐M. , Cistarelli L. , Millan M. J.
Published: July 2010

Abstract: A serotonin (5‐HT)1A receptor partial agonist, buspirone, potentiates the clinical antidepressant properties of 5‐HT reuptake inhibitors (SSRIs). Herein, we examined the interaction of buspirone with two SSRIs, duloxetine and fluoxetine, on extra‐cellular levels of 5‐HT, dopamine (DA), and noradrenaline (NAD) in single dialysate samples of freely moving rats. Duloxetine (5.0 mg/kg, s.c.) and fluoxetine (10.0 mg/kg, s.c.) increased dialysate levels of DA (65 and 60% vs. basal values, respectively), NAD (400 and 90%, respectively), and 5‐HT (130 and 110%, respectively) in the frontal cortex (FCX). Buspirone (2.5 mg/kg, s.c.) similarly elevated levels of DA (100%) and NAD (160%) but reduced those of 5‐HT (−50%). Administered with buspirone, the ability of duloxetine and fluoxetine to increase 5‐HT levels was transiently inhibited (over 60 min), although by the end of sampling (180 min) their actions were fully expressed. In contrast, buspirone markedly and synergistically facilitated the elevation in DA levels elicited by duloxetine (550%) and fluoxetine (240%). Furthermore, buspirone potentiated the induction of NAD levels by duloxetine (750%) and fluoxetine (350%). These data suggest that a reinforcement in the influence of SSRIs on DA and, possibly, NAD but not 5‐HT release in FCX may contribute to their increased antidepressant activity in the presence of buspirone. More generally, they support the hypothesis that a reinforcement in dopaminergic transmission in the FCX contributes to the actions of SSRIs and other antidepressant drugs.
A Review of Phylogenetic and Metabolic Relationships Between the Acylamino Acids, N‐Acetyl‐l‐Aspartic Acid and N‐Acetyl‐l‐Histidine, in the Vertebrate Nervous System
Authors: Baslow Morris H.
Published: July 2010

Abstract: N‐Acetyl‐l‐histidine (NAH) and N‐acetyl‐l‐aspartic acid (NAA) are major constituents of vertebrate brain and eye with distinct phylogenetic distributions. They are characterized by high tissue concentrations, high tissue/extracellular fluid gradients, and a continuous regulated efflux into the extracellular fluid. As a result of parallel investigations over the past three decades, evidence has accumulated that suggests that the metabolism of NAA in the CNS of both homeothermic and poikilothermic vertebrates and the metabolism of NAH in the CNS of poikilothermic vertebrates are related. Tissue distribution and concentrations are similar, as well as timing of appearance during embryological development and their synthetic and degradative biochemistry. Both amino acids appear to be involved in a rapid tissue‐to‐fluid‐space cycling phenomenon across a membrane. Evidence accumulating for each amino acid suggests a dynamic and important role in the CNS and the eye of vertebrates. A genetic disease in humans, Canavan's disease, is associated with NAA aciduria and aspartoacylase deficiency with concomitant accumulation of NAA and a spongy degeneration of the brain. In this article, evidence linking NAA and NAH metabolism is reviewed, and the hypothesis that NAA and NAH complement each other and are metabolic analogues involved with membrane transport is developed. Their enzyme systems also appear to exhibit plasticity in relation to osmoregulatory forces on an evolutionary time scale, with an apparent interface at the fish‐tetrapod boundary.
Evidence that the Early Loss of Membrane Protein Kinase C Is a Necessary Step in the Excitatory Amino Acid‐Induced Death of Primary Cortical Neurons
Authors: Durkin J. P. , Tremblay R. , Chakravarthy B. , Mealing G. , Morley P. , Small D. , Song D.
Published: July 2010

Abstract: A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15–20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20–50 µM glutamate for 15 min was neurotoxic and induced a rapid (∼1–2 h) Ca2+‐dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to >800 µM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription‐polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2+ concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptor on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (<20 µM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 µM calphostin C, 5 µM chelerythrine, or chronic exposure to 100 nM PMA. A 15‐min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate‐mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate‐mediated neuronal death.
Ciliary Neurotrophic Factor Activates JAK/Stat Signal Transduction Cascade and Induces Transcriptional Expression of Glial Fibrillary Acidic Protein in Glial Cells
Authors: Kahn M. A. , Huang C. J. , Caruso A. , Barresi V. , Nazarian R. , Condorelli D. F. , De Vellis J.
Published: July 2010

Abstract: In recent reports, ciliary neurotrophic factor (CNTF) has been implicated as an injury factor involved in regulating astrogliosis in the CNS. In this study, we used a rat oligodendroglial progenitor cell line that is highly responsive to CNTF to examine CNTF‐induced alterations that may play a role in activation of the glial fibrillary acidic protein (GFAP) gene. We determined that CNTF induces the transient translocation of Stat1α/p91 to the nucleus. This nuclear translocation was followed by GFAP promoter activation and an up‐regulation of GFAP mRNA and protein. Levels of CNTF‐α receptor mRNA, however, were unaffected by addition of the ligand. Transfection studies using an upstream 5′‐flanking, 1.9‐kb rat GFAP promoter linked to a luciferase reporter gene revealed CNTF‐induced transcriptional activation within 1 h of ligand exposure. Moreover, serial‐deleted constructs identified a distal (−1,857 to −1,546 bp) and a proximal (−384 to −106 bp) region as being important for CNTF‐induced GFAP promoter activation. These two regions showed a strong degree of overlap for CNTF‐ and serum‐induced activation of the GFAP gene. Analysis of the two regions revealed several cis‐elements that are thought to be involved in GFAP regulation and/or the regulation of other genes by members of the interleukin‐6 family of cytokines. Moreover, we are the first to report the presence of several putative CNTF‐responsive elements within our identified distal and proximal regions in the GFAP gene promoter.
Cyclic GMP Inhibits a Pharmacologically Distinct Na+/H+ Exchanger Variant in Cultured Rat Astrocytes via an Extracellular Site of Action
Authors: Touyz Rhian M. , Picard Sylvie , Schiffrin Ernesto L. , Deschepper Christian F.
Published: July 2010

Abstract: There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP‐inducing C‐type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pHi). To examine whether this effect was due to inhibition of an Na+/H+ exchanger (NHE), we acidified cells by replacing extracellular Na+ by choline and examined the kinetics of the pHi recovery that occurred on reintroduction of Na+ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na+‐dependent pHi recovery, even in the nominal absence of CO2/HCO3−. This indicated that CNP inhibited the activity of an exchanger that was Na+‐dependent, HCO3−‐independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium‐dependent pHi recovery, but the native nucleotide was as potent as membrane‐permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na+‐dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pHi transients have been shown to modulate several important astrocytic functions.
l‐trans‐Pyrrolidine‐2,4‐Dicarboxylic Acid‐Evoked Striatal Glutamate Levels Are Attenuated by Calcium Reduction, Tetrodotoxin, and Glutamate Receptor Blockade
Authors: Rawls Scott M. , McGinty Jacqueline F.
Published: July 2010

Abstract: l‐trans‐Pyrrolidine‐2,4‐dicarboxylic acid (l‐trans‐PDC) reverses plasma membrane glutamate transporters and elevates extracellular glutamate levels in vivo. We investigated the possibility that l‐trans‐PDC‐stimulated glutamate levels are mediated partially by increases in transsynaptic activity. Therefore, the degree to which l‐trans‐PDC‐evoked glutamate levels depend on calcium, sodium‐channel activation, and glutamate‐receptor activation was investigated by infusing via reverse microdialysis (a) 0.1 mM calcium, (b) 1 µM tetrodotoxin, a selective blocker of voltage‐dependent sodium channels, (c) R(−)‐3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid (CPP), a selective NMDA‐receptor antagonist, or (d) LY293558, a selective α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate antagonist. In separate experimental groups, l‐trans‐PDC‐evoked glutamate levels were reduced significantly by 55% in the presence of 0.1 mM calcium and by 46% in the presence of tetrodotoxin. Additionally, CPP and LY293558 significantly attenuated l‐trans‐PDC‐evoked glutamate levels without altering basal glutamate levels. These data suggest that glutamate transporter reversal by l‐trans‐PDC initially elevates extracellular glutamate levels enough to stimulate postsynaptic glutamate receptors within the striatum. It is proposed that glutamate‐receptor stimulation activates a positive feedback loop within the basal ganglia, leading to further glutamate release from corticostriatal and thalamostriatal afferents. Therefore, either extracellular striatal calcium reduction or tetrodotoxin perfusion leads to decreased action potential‐dependent glutamate release from these terminals. In addition, blocking glutamate receptors directly reduces medium spiny neuronal firing and indirectly attenuates corticostriatal and thalamostriatal activity, resulting in an overall depression of l‐trans‐PDC‐stimulated glutamate levels.
Alterations in Glutamate Transporter Protein Levels in Kindling‐Induced Epilepsy
Authors: Miller Heather Prince , Levey Allan I. , Rothstein Jeffrey D. , Tzingounis Anastassios V. , Conn P. Jeffrey
Published: July 2010

Abstract: There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high‐affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with seizures. To date, three distinct Na+‐dependent glutamate transporters have been cloned from rat and rabbit: GLT‐1, GLAST, and EAAC‐1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala‐kindled rats. Levels of GLT‐1, GLAST, and EAAC‐1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype‐specific antibodies. GLAST protein was down‐regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC‐1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. No changes in GLT‐1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.
Matrix Metalloproteinases in Dog Brains Exhibiting Alzheimer‐Like Characteristics
Authors: Lim Giselle P. , Russell Michael J. , Cullen Michael J. , Tökés Zoltán A.
Published: July 2010

Abstract: We have previously reported that the amount of the neuronal matrix metalloproteinase (MMP) MMP‐9, capable of cleaving β‐amyloid1–40 predominantly at Leu34‐Met35, is increased in a latent form in hippocampal specimens from AD patients and have suggested that the lack of activation of this enzyme may contribute to the deposition of β‐amyloid in plaques. The current study addresses whether similar matrix proteinases are detectable in amyloid‐positive and ‐negative brain specimens of aged beagles. Using quantitative zymography, three major neutral proteinases with molecular masses of 60, 95, and 280 kDa were readily detected. These enzymes have the characteristics of MMPs because they were inhibited by EDTA and 1,10‐phenanthroline, and their activities were restored by addition of both Ca2+ and Zn2+. The 95‐ and 280‐kDa proteinases cross‐reacted with specific monoclonal antibodies to human MMP‐9 (gelatinase B; EC 3.4.24.35). Canine MMP‐9 was latent because activation by organomercurial treatment resulted in a characteristic decrease in molecular mass. Statistical analysis revealed no difference in the 60‐kDa proteinase activity in amyloid‐positive and ‐negative brain specimens. However, significantly increased amounts of latent MMP‐9 were observed in amyloid‐positive brain specimens (p≤ 0.05) compared with amyloid‐negative brain specimens. The observations document that changes in MMP‐9 expression in amyloid‐positive beagle brains are similar to those reported in the human Alzheimer's disease hippocampus and suggest the possibility that insufficient activation of MMP‐9 may contribute to β‐amyloid accumulation, a hypothesis that needs to be further investigated.
Role of Calpain‐ and Interleukin‐1β Converting Enzyme‐Like Proteases in the β‐Amyloid‐Induced Death of Rat Hippocampal Neurons in Culture
Authors: Jordán Joaquín , Galindo María F. , Miller Richard J.
Published: July 2010

Abstract: We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by β‐amyloid(Aβ) (25–35). Both Aβ(25–35)‐ and staurosporine‐induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA‐induced death appeared more complex. Two irreversible inhibitors of the interleukin‐1β converting enzyme (ICE) and related proteases, Z‐Val‐Ala‐Asp‐CH2F and acetyl‐Tyr‐Val‐Ala‐Asp‐chloromethyl ketone, blocked neuronal death produced by Aβ(25–35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium‐regulated protease calpain, also inhibited death induced by all agents. Aβ(25–35) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA‐induced death. These results support the role of apoptosis in neuronal death due to Aβ(25–35) treatment and also suggest a role for calcium‐regulated proteases in this process.
Neuronal Regulation of Interleukin 6 Secretion in Murine Spleen: Adrenergic and Opioidergic Control
Authors: Straub Rainer H. , Herrmann Markus , Berkmiller Gebhard , Frauenholz Thomas , Lang Bernhard , Schölmerich Jürgen , Falk Werner
Published: July 2010

Abstract: The PNS was anticipated to be involved in the modulation of immune responses. To study aspects of this neuronal‐immune communication, a recently developed tissue slice method was used to study the effects of adrenergic and opioidergic transmitters on interleukin 6 (IL‐6) secretion in the spleen. The α2‐adrenergic agonist p‐aminoclonidine (10−7M) inhibited IL‐6 secretion (control vs. p‐aminoclonidine, 100.0 ± 4.76 vs. 59.3 ± 6.6% of control values; p < 0.001). The α1‐adrenergic agonist methoxamine (10−8M) also inhibited IL‐6 secretion (100.0 ± 4.8 vs. 71.5 ± 3.8%; p < 0.001). The endogenous opioids β‐endorphin (10−10M), methionine‐enkephalin (10−9M), and leucine‐enkephalin (10−9M) inhibited IL‐6 secretion as well (p = 0.0051, p = 0.0337, and p = 0.0226, respectively). Electrical stimulation of spleen slices inhibited IL‐6 secretion (100.0 ± 4.3 vs. 56.7 ± 4.6% of control values; p < 0.001). The involvement of α‐adrenergic and opioidergic molecules in this electrically induced inhibition was shown by the use of antagonists. Electrical inhibition of IL‐6 secretion was attenuated by phentolamine (10−7M; p = 0.0345), by naloxone (10−6M; p = 0.0046), by cyprodime (10−8M; p = 0.0014), and by the combination of cyprodime (10−7M) plus phentolamine (10−8M; p < 0.0001). We conclude from the complementary studies that the inhibition of IL‐6 secretion induced by electrical pulses was mostly mediated by α‐adrenergic and μ‐opioidergic endogenous transmitters.
Cytoskeletal Changes in the Brains of Mice Lacking Calcineurin Aα
Authors: Kayyali Usamah S. , Zhang Wei , Yee Ann G. , Seidman J. G. , Potter Huntington
Published: July 2010

Abstract: Hyperphosphorylated τ, the major component of the paired helical filaments of Alzheimer's disease, was found to accumulate in the brains of mice in which the calcineurin Aα gene was disrupted [calcineurin Aα knockout (CNAα−/−)]. The hyperphosphorylation involved several sites on τ, especially the Ser396 and/or Ser404 recognized by the PHF‐1 monoclonal antibody. The increase in phosphorylated τ content occurred primarily in the mossy fibers of the CNAα−/− hippocampus, which contained the highest level of calcineurin in brains of wild‐type mice. The CNAα−/− mossy fibers also contained less neurofilament protein than normal, although the overall level of neurofilament phosphorylation was unchanged. In the electron microscope, the mossy fibers of CNAα−/− mice exhibited abnormalities in their cytoskeleton and a lower neurofilament/microtubule ratio than those of wild‐type animals. These findings indicate that hyperphosphorylated τ can accumulate in vivo as a result of reduced calcineurin activity and is accompanied by cytoskeletal changes that are likely to have functional consequences on the affected neurons. The CNAα−/− mice were found in a separate study to have deficits in learning and memory that may result in part from the cytoskeletal changes in the hippocampus.
Ca2+ and Reactive Oxygen Species in Staurosporine‐Induced Neuronal Apoptosis
Authors: Prehn J. H. M. , Jordán J. , Ghadge G. D. , Preis E. , Galindo M. F. , Roos R. P. , Krieglstein J. , Miller R. J.
Published: July 2010

Abstract: Staurosporine (0.03–0.5 µM) induced a dose‐dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24‐h pretreatments with the protein synthesis inhibitor cycloheximide (1 µM) or the cell cycle inhibitor mimosine (100 µM). To investigate the role of Ca2+ and reactive oxygen species in staurosporine‐induced neuronal apoptosis, we overexpressed calbindin D28K, a Ca2+ binding protein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus‐mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a β‐galactosidase overexpressing adenovirus. Staurosporine‐induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage‐sensitive Ca2+ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1–10 µM) nor the NMDA receptor antagonist dizocilpine (MK‐801; 1 µM) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U‐74500A (1–10 µM) and N‐acetylcysteine (100 µM) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca2+ and reactive oxygen species in staurosporine‐induced neuronal apoptosis.
Basic Amino Acids at the C‐Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin‐B Receptors
Authors: Wang Hung‐Li
Published: July 2010

Abstract: In common with other Gq protein‐coupled receptors, the third intracellular loop of the cholecystokinin‐B (CCK‐B) receptor contains three basic amino acids (K333/K334/R335) at the C‐terminal segment. To determine the importance of these conserved basic residues in Gq‐protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS‐7 cells and recording Ca2+‐activated Cl− currents from Xenopus oocytes. Site‐directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK‐B receptors were expressed in COS‐7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK‐8) failed to induce inositol phosphate formation in COS‐7 cells and evoke Ca2+‐activated Cl− currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single‐point mutations resulted in a significant reduction in CCK‐8‐induced inositol phosphate formation and CCK‐8‐activated Ca2+‐dependent Cl− currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK‐8‐simulated inositol phosphate formation and the amplitude of CCK‐8‐evoked Ca2+‐dependent Cl− currents. Radioligand‐binding studies showed that the above‐mentioned mutations did not affect the affinity for CCK‐8 and receptor expression level in COS‐7 cells. These findings suggest that basic amino acids at the C‐terminus of the third cytoplasmic loop are required for the signal transduction by CCK‐B receptors.
Regulation of Myelin‐Associated Glycoprotein Binding by Sialylated Cis‐Ligands
Authors: Tropak M. B. , Roder J. C.
Published: July 2010

Abstract: Myelin‐associated glycoprotein (MAG) and Schwann cell myelin protein (SMP) are highly glycosylated members of a newly defined family of cell adhesion molecules belonging to the immunoglobulin superfamily that recognize terminal sialic acid residues on N‐ and O‐linked oligosaccharides. The importance of the N‐linked oligosaccharides on MAG were determined by removal of the eight predicted carbohydrate addition sites by site‐directed mutagenesis. The results suggest that all eight N‐linked glycosylation sites are utilized in COS cells. N‐linked glycosylation does not appear to be required for sialic acid‐dependent MAG binding to erythrocytes. However, N‐linked glycosylation of MAG does play a role in the proper folding of MAG. It was also shown that sialylation in the host cell expressing MAG and SMP could inhibit binding to erythrocytes. The degree to which SMP and MAG erythrocyte binding was affected by sialylation in the host cell was dependent on (a) the level at which MAG was expressed on the surface on the host cell and (b) the presence of MAG ligands on the host cell. The data suggest that cis‐ligands on the host cell compete with trans‐ligands on the target cell for the binding site(s) on MAG.
An Etiological Role of Amyloidogenic Carboxyl‐Terminal Fragments of the β‐Amyloid Precursor Protein in Alzheimer's Disease
Authors: Suh Yoo‐Hun
Published: July 2010

Abstract: Amyloid β protein (Aβ), 39–43 amino acids long, is the principal constituent of the extracellular amyloid deposits in brain that are characteristic of Alzheimer's disease (AD). Several lines of evidence indicate that Aβ may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of Aβ and the development of the disease. Thus, Aβ may not be the sole active fragment of β‐amyloid precursor protein (βAPP) in the neurotoxicity associated with AD. Consequently, the possible effects of other cleaved products of βAPP need to be explored. The recent concentration on other potentially amyloidogenic products of βAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl‐terminal (CT) fragments of βAPP. This review discusses a possible etiological role of CT fragments of βAPP in AD.
Temperature‐Sensitive Expression of Drosophila Neuronal Nicotinic Acetylcholine Receptors
Authors: Lansdell Stuart J. , Schmitt Bertram , Betz Heinrich , Sattelle David B. , Millar Neil S.
Published: July 2010

Abstract: Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37°C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR β subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full‐length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature‐sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits.
PTP NE‐6: A Brain‐Enriched Receptor‐Type Protein Tyrosine Phosphatase with a Divergent Catalytic Domain
Authors: Fitzgerald Laura Rydelek , Walton Kevin M. , Dixon Jack E. , Largent Brian L.
Published: July 2010

Abstract: A receptor‐type protein tyrosine phosphatase, PTP NE‐6, was identified from rat olfactory epithelial cDNA and cloned from a rat brain cDNA library. PTP NE‐6 mRNA is abundant in brain and expressed at lower levels in olfactory tissue and adrenal gland. In situ hybridization demonstrates that PTP NE‐6 mRNA is expressed throughout the brain, with highest levels in the medial habenula and at intermediate levels in layer IV of cortex, medial geniculate nucleus, inferior colliculus, hypothalamus, and thalamus. The predicted amino acid sequence demonstrates that PTP NE‐6 contains a single catalytic domain that diverges from the consensus protein tyrosine phosphatase catalytic domain by expressing an aspartate instead of the conserved alanine residue in the catalytic site. Recombinantly expressed PTP NE‐6 does not exhibit detectable phosphatase activity. Upon mutation of the aspartate to the consensus alanine, phosphatase activity toward p‐nitrophenyl phosphate is observable with a kcat value of 3.7 s−1 and a Km of 980 µM. These data demonstrate that the inactivity of native PTP NE‐6 toward p‐nitrophenyl phosphate is due to the divergent aspartate in the catalytic site and not to variant amino acids within the phosphatase domain.
NMDA Receptor‐Mediated Neurotoxicity: A Paradoxical Requirement for Extracellular Mg2+ in Na+/Ca2+‐Free Solutions in Rat Cortical Neurons In Vitro
Authors: Hartnett K. A. , Stout A. K. , Rajdev S. , Rosenberg P. A. , Reynolds I. J. , Aizenman E.
Published: July 2010

Abstract: Accumulation of intracellular Ca2+ is known to be critically important for the expression of NMDA receptor‐mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg2+ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg2+ content independently of Ca2+ to investigate the potential role of Mg2+ in excitotoxicity in rat cortical neurons in vitro. In Ca2+‐free solutions in which the Na+ was replaced by N‐methyl‐d‐glucamine or Tris (but not choline), which also contained 9 mM Mg2+, exposure to 100 µM glutamate or 200 µM NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg2+ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg2+‐dependent NMDA receptor‐mediated toxicity. Thus, the increase in intracellular Mg2+ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.
Modulation of Endogenous Antioxidant Enzymes by Nitric Oxide in Rat C6 Glial Cells
Authors: Dobashi Kazushige , Pahan Kalipada , Chahal Amarjit , Singh Inderjit
Published: July 2010

Abstract: To understand the possible mechanism of nitric oxide (NO)‐mediated cytotoxicity, we investigated the effect of NO on the endogenous antioxidant enzymes (AOEs) catalase, glutathione peroxidase (GPX), and CuZn‐ and Mn‐superoxide dismutases (SODs) in rat C6 glial cells under conditions in which these cells expressed oligodendrocyte‐like properties as evidenced by the expression of 2′,3′‐cyclic‐nucleotide 3′‐phosphohydrolase. The 24‐h treatment with S‐nitroso‐N‐acetylpenicillamine (SNAP), a NO donor, decreased the activities and the protein levels of catalase, GPX, and Mn‐SOD in a dose‐dependent manner. Alternatively, the activity and the protein level of CuZn‐SOD were increased. 2‐Phenyl‐4,4, 5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO), a NO scavenger, blocked the effect of SNAP. Moreover, the treatment of C6 cells with sodium nitroprusside, another NO donor, or with a combination of lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ), which induce excessive production of NO, also significantly modulated the AOE activities in a manner similar to that seen with SNAP treatment. The compounds/enzymes that inhibit the production of NO (e.g., N‐nitro‐l‐arginine methyl ester hydrochloride, arginase, and PTIO) blocked the effects of LPS and IFN‐γ on the activities of AOEs. Treatment with SNAP and a combination of LPS and IFN‐γ also modulated the mRNA levels of AOEs, parallel to the changes in their protein levels and activities, except for Mn‐SOD where the combination of LPS and IFN‐γ markedly stimulated the mRNA expression. In spite of the stimulation of mRNA level, LPS and IFN‐γ significantly inhibited the activity of Mn‐SOD within the first 24 h of incubation; however, Mn‐SOD activity gradually increased with the increase in time of incubation. These results suggest that alterations in the status of AOEs by NO may be the basis of NO‐induced cytotoxicity in disease states associated with excessive NO production.
Expression of Krox Proteins During Differentiation of the O‐2A Progenitor Cell Line CG‐4
Authors: Sock Elisabeth , Leger Hubert , Kuhlbrodt Kirsten , Schreiber Jörg , Enderich Janna , Richter‐Landsberg Christiane , Wegner Michael
Published: July 2010

Abstract: Krox proteins are important regulators of development and terminal differentiation. Using the rat glial progenitor cell line CG‐4 as a model system for oligodendrocyte differentiation, we show that on the RNA level Krox‐24 is the predominant member of the Krox family in these cells. Similar results were also obtained on the protein level as the major Krox protein from CG‐4 cell extracts reacted specifically with an antibody against Krox‐24. Whereas Krox‐24 RNA and protein were abundant in undifferentiated CG‐4 cells, a dramatic decrease in expression was detected after a 3–5‐day period of differentiation during which we observed a reciprocal increase in the levels of myelin basic protein expression. Importantly, regulation of Krox‐24 expression was very similar in CG‐4 cells and primary oligodendrocyte cultures. When expression of Krox‐24 in differentiating CG‐4 cells was followed on a closer time scale, we observed a sharp and transient increase in Krox‐24 RNA, protein, and DNA binding activity immediately after the onset of differentiation followed by an equally rapid decrease. This expression pattern implicates Krox‐24 both in maintenance of the undifferentiated state and in the immediate early phase of differentiation of CG‐4 cells and possibly oligodendrocytes.
Muscarinic Modulation of Striatal Dopamine, Glutamate, and GABA Release, as Measured with In Vivo Microdialysis
Authors: Smolders Ilse , Bogaert Liesbeth , Ebinger Guy , Michotte Yvette
Published: July 2010

Abstract: Intrastriatal microdialysis was used to administer muscarinic drugs in freely moving rats for 40 min at a flow rate of 2 µl/min. Administration of the nonselective agonist pilocarpine at 10 mM increased striatal dopamine release and decreased extracellular GABA and glutamate overflow. Perfusion with the muscarinic M2 antagonist methoctramine at 75 µM increased extracellular dopamine and glutamate concentrations but exerted no changes on extracellular GABA levels. Intrastriatal administration of the M1 antagonist pirenzepine at 0.05 µM decreased extracellular dopamine overflow. Application of pirenzepine (0.05 and 5 µM) exerted no effects on the measured GABA or glutamate levels. There are thus important differences in applied doses of muscarinic drugs needed to obtain modulatory effects. High doses of agonists are probably needed to superimpose on the background of tonic influences of striatal acetylcholine, whereas antagonists can block the receptors in small doses. We further suggest that M1 receptors might tonically facilitate striatal dopamine release, that M2 receptors might tonically inhibit striatal glutamate efflux, and that acetylcholine does not exert tonic effects on striatal GABA release. The link with the pilocarpine animal model for temporal lobe epilepsy will be discussed.