bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol Exposure Affects Glial Fibrillary Acidic Protein Gene Expression and Transcription During Rat Brain Development
Authors: Vallés S. , Pitarch J. , Renau‐Piqueras J. , Guerri C.
Published: July 2010

Abstract: Exposure to ethanol during fetal development reduces the astroglial‐specific marker glial fibrillary acidic protein (GFAP) and its mRNA levels in brains of fetal rats and in radial glia in primary culture, affecting the proliferation and differentiation of astrocytes. The objectives of this study were to evaluate the possible effect of ethanol on GFAP mRNA levels in astrocytes and to investigate the molecular mechanism(s) involved in ethanol‐induced changes in GFAP expression by analyzing the GFAP transcription rate, GFAP mRNA stability, and GFAP DNA methylation. We show here that prenatal exposure to ethanol reduces significantly GFAP immunoreactivity and its mRNA levels in both astrocytes in primary culture and brains of pups from alcohol‐fed mothers. Runoff experiments from nuclei of astrocytes indicate that ethanol exposure decreases GFAP transcription rate significantly and reduces GFAP mRNA stability slightly. DNA methylation analysis indicates that prenatal ethanol exposure induces a hypermethylated state of the GFAP DNA in fetal brains. Methylation‐mediated repression of GFAP transcription could be a mechanism involved in ethanol‐induced reduction of GFAP expression. Ethanol‐induced alterations in GFAP expression and astroglial development may underlie the CNS dysfunctions observed after prenatal alcohol exposure.
Biochemical Characterization and Histochemical Localization of Nitric Oxide Synthase in the Nervous System of the Snail, Helix pomatia
Authors: Huang Shile , Kerschbaum Hubert H. , Engel Edwin , Hermann Anton
Published: July 2010

Abstract: Nitric oxide synthase (NOS) in the snail Helix pomatia was characterized by biochemical and molecular biological techniques and localized by histochemical methods. Central ganglia contained particulate paraformaldehyde‐sensitive and cytosolic paraformaldehyde‐insensitive NADPH‐diaphorase. The cytosolic NADPH‐diaphorase activity coeluted with NOS activity. The activity of NOS was dependent on Ca2+ and NADPH and was inhibited by NG‐nitro‐l‐arginine (l‐NNA). Proteins purified by 2′,5′‐ADP affinity chromatography were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and migrated at 150, 60, 40, and 30 kDa. An antibody to mammalian NOS exclusively labeled the 60‐kDa protein. Characterization of the cDNA of the corresponding 60‐kDa NOS‐immunoreactive protein revealed no sequence homology with any known NOS isoform. The recombinant protein exhibited Ca2+‐ and NADPH‐dependent NOS activity, which was partially inhibited by EGTA and l‐NNA. Histochemistry showed NADPH‐diaphorase activity in discrete regions of the central and peripheral nervous system. About 60% of the NADPH‐diaphorase‐positive neurons colocalize with immunoreactive material detected by antibodies to mammalian NOS. Comparison of organs showed the highest NADPH‐diaphorase activity in the nervous system, whereas moderate activity was present in muscle tissue, digestive tract, and gonads. Our study suggests the presence of NOS and a putative NOS‐associated/regulating protein in mollusk nervous tissue.
Presenilins, the Endoplasmic Reticulum, and Neuronal Apoptosis in Alzheimer's Disease
Authors: Mattson Mark P. , Guo Qing , Furukawa Katsutoshi , Pedersen Ward A.
Published: July 2010

Abstract: Many cases of autosomal dominant inherited forms of early‐onset Alzheimer's disease are caused by mutations in the genes encoding presenilin‐1 (PS‐1; chromosome 14) and presenilin‐2 (PS‐2; chromosome 1). PSs are expressed in neurons throughout the brain wherein they appear to be localized primarily to the endoplasmic reticulum (ER) of cell bodies and dendrities. PS‐1 and PS‐2 show high homology and are predicted to have eight transmembrane domains with the C terminus, N terminus, and a loop domain all on the cytosolic side of the membrane; an enzymatic cleavage of PSs occurs at a site near the loop domain. The normal function of PSs is unknown, but data suggest roles in membrane trafficking, amyloid precursor protein processing, and regulation of ER calcium homeostasis. Homology of PSs to the C. elegans gene sel‐12, which is involved in Notch signaling, and phenotypic similarities of PS‐1 and Notch knockout mice suggest a developmental role for PSs in the nervous system. When expressed in cultured cells and transgenic mice, mutant PSs promote increased production of a long form of amyloid β‐peptide (Aβ1‐42) that may possess enhanced amyloidogenic and neurotoxic properties. PS mutations sensitize cultured neural cells to apoptosis induced by trophic factor withdrawal, metabolic insults, and amyloid β‐peptide. The mechanism responsible for the proapoptotic action of mutant PSs may involve perturbed calcium release from ER stores and increased levels of oxidative stress. Recent studies of apoptosis in many different cell types suggest that ER calcium signaling can modulate apoptosis. The evolving picture of PS roles in neuronal plasticity and Alzheimer's disease is bringing to the forefront the ER, an organelle increasingly recognized as a key regulator of neuronal plasticity and survival.
Bcl‐2 Protects Isolated Plasma and Mitochondrial Membranes Against Lipid Peroxidation Induced by Hydrogen Peroxide and Amyloid β‐Peptide
Authors: Bruce‐Keller Annadora J. , Begley James G. , Fu Weiming , Butterfield D. Allan , Bredesen Dale E. , Hutchins James B. , Hensley Kenneth , Mattson Mark P.
Published: July 2010

Abstract: The bcl‐2 protooncogene product possesses antiapoptotic properties in neuronal and nonneuronal cells. Recent data suggest that Bcl‐2's potency as a survival factor hinges on its ability to suppress oxidative stress, but neither the subcellular site(s) nor the mechanism of its action is known. In this report electron paramagnetic resonance (EPR) spectroscopy analyses were used to investigate the local effects of Bcl‐2 on membrane lipid peroxidation. Using hydrogen peroxide (H2O2) and amyloid β‐peptide (Aβ) as lipoperoxidation initiators, we determined the loss of EPR‐detectable paramagnetism of nitroxyl stearate (NS) spin labels 5‐NS and 12‐NS. In intact cell preparations and postnuclear membrane fractions, Aβ and H2O2 induced significant loss of 5‐NS and 12‐NS signal amplitude in control PC12 cells, but not PC12 cells expressing Bcl‐2. Cells were subjected to differential subcellular fractionation, yielding preparations of plasma membrane and mitochondria. In preparations derived from Bcl‐2‐expressing cells, both fractions contained Bcl‐2 protein. 5‐NS and 12‐NS signals were significantly decreased following Aβ and H2O2 exposure in control PC12 mitochondrial membranes, and Bcl‐2 largely prevented these effects. Plasma membrane preparations containing Bcl‐2 were also resistant to radical‐induced loss of spin label. Collectively, our data suggest that Bcl‐2 is localized to mitochondrial and plasma membranes where it can act locally to suppress oxidative damage induced by Aβ and H2O2, further highlighting the important role of lipid peroxidation in apoptosis.
Inhibition of Depolarization‐Induced Nitric Oxide Synthase Activation by NS‐7, a Phenylpyrimidine Derivative, in Primary Neuronal Culture
Authors: Tatsumi S. , Itoh Y. , Ma F.‐H. , Higashira H. , Ukai Y. , Yoshikuni Y. , Kimura K.
Published: July 2010

Abstract: Neuronal nitric oxide synthase (NOS) is considered to be involved in the pathogenesis of ischemic brain damage. In the present study, the effect of a novel neuroprotective phenylpyrimidine derivative, 4‐(4‐fluorophenyl)‐2‐methyl‐6‐(5‐piperidinopentyloxy)pyrimidine hydrochloride (NS‐7), on depolarization‐stimulated NOS activity was examined in cultured neurons of mouse cerebral cortex. Various depolarizing stimuli such as veratridine, KCl, and N‐methyl‐d‐aspartate increased the NOS activity determined by cyclic GMP formation. NS‐7 concentration‐dependently inhibited both the veratridine‐ and KCl‐induced NOS activation with IC50 values of 9.3 and 9.6 µM, respectively. The reversal of KCl‐evoked NOS activity by NS‐7 was also observed under blockade of both ionotropic glutamate receptors and the Na+ channel with MK‐801, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and tetrodotoxin. In contrast, NS‐7, even at 100 µM, did not affect N‐methyl‐d‐aspartate‐stimulated NOS activity, nor did it have any influence on NOS activity determined in the soluble fraction of rat hippocampus. Because NS‐7 has already been shown to block both Na+ and Ca2+ channels, the present findings suggest that this compound inhibits depolarization‐induced NOS activation by reducing Ca2+ influx through blockade of Na+ and Ca2+ channels in primary neuronal culture.
The Effect of Magnesium on Oxidative Neuronal Injury In Vitro
Authors: Regan Raymond F. , Jasper Edward , Guo Yaping , Panter S. Scott
Published: July 2010

Abstract: The effect of magnesium on the oxidative neuronal injury induced by hemoglobin was assessed in murine cortical cell cultures. Exposure to 5 µM hemoglobin in physiologic (1 mM) magnesium for 26 h resulted in the death of about one‐half the neurons and a sixfold increase in malondialdehyde production; glia were not injured. Increasing medium magnesium to 3 mM reduced neuronal death by about one‐half and malondialdehyde production by about two‐thirds; neuronal death and lipid peroxidation were approximately doubled in 0.3 mM magnesium. Comparable results were observed in spinal cord cultures. The NMDA antagonist MK‐801 weakly attenuated hemoglobin neurotoxicity in low‐magnesium medium, but tended to potentiate injury in physiologic magnesium. Incubation in low‐magnesium medium alone for 24 h reduced cellular glutathione by ∼50% in mixed neuronal and glial cultures but by only 10% in pure glial cultures. The iron‐dependent oxidation of phosphatidylethanolamine liposomes was attenuated in a concentration‐dependent fashion by 2.5–10 mM magnesium; a similar effect was provided by 0.01–0.1 mM cobalt. However, oxidation was weakly enhanced by 0.5–1 mM magnesium. These results suggest that the vulnerability of neurons to iron‐dependent oxidative injury is an inverse function of the extracellular magnesium concentration. At high concentrations, magnesium inhibits lipid peroxidation directly, perhaps by competing with iron for phospholipid binding sites. At low concentrations, enhancement of cell death may be due to the combined effect of increased NMDA receptor activity, glutathione depletion, and direct potentiation of lipid peroxidation.
In Vivo and In Vitro Characterization of the Sequence Requirement for Oligomer Formation of Ca2+/Calmodulin‐Dependent Protein Kinase IIα
Authors: Shen Kang , Meyer Tobias
Published: July 2010

Abstract: Ca2+/calmodulin‐dependent protein kinase II (CaMK II) is a multifunctional serine/threonine protein kinase that regulates ion channels, metabolic enzymes, cytoskeletal proteins, and possibly transcription factors. CaMK II holoenzymes have been shown to be large oligomers built from eight or more subunits. The oligomeric structure of CaMK II is required for a critical trans‐autophosphorylation step by which each subunit traps bound calmodulin and renders the enzyme partially active in the absence of Ca2+/calmodulin. Here we define a minimal C‐terminal domain of αCaMK II that is necessary and sufficient for oligomerization. The oligomeric structure of αCaMK II deletion mutants was investigated in vitro by gel filtration chromatography and in living cells by measuring nuclear exclusion and diffusion coefficients of green fluorescent protein‐tagged mutants. These studies showed that a C‐terminal region of αCaMK II of 135 amino acids is sufficient for oligomer formation. Oligomer formation was abolished by further C‐terminal and N‐terminal deletions, indicating that the same region is not only sufficient but also necessary for oligomerization. Thus, an oligomerization domain of only 15 kDa is sufficient to build the circular structure of CaMK II holoenzyme.
Ca2+/Calmodulin‐Dependent Transcriptional Activation of δ‐Opioid Receptor Gene Expression Induced by Membrane Depolarization in NG108‐15 Cells
Authors: Buzas Beata , Rosenberger John , Cox Brian M.
Published: July 2010

Abstract: Regulation of gene expression is one of the mechanisms by which neuronal activity elicits long‐term changes in neuronal phenotype and function. Although activity‐dependent induction of immediate‐early genes has been extensively studied, much less is known about the late‐response genes. We have investigated the activity‐dependent regulation of δ‐opioid receptor (DOR) mRNA levels in NG108‐15 cells. Transsynaptic activation was mimicked by depolarization with 55 mM KCl or veratridine. Both treatments lead to a time‐dependent increase of DOR mRNA levels. Ca2+ entry through L‐type voltage‐dependent Ca2+ channels activated by depolarization appears to be involved, because L‐type channel blockers reduced the induction of DOR expression. Ca2+ binding to calmodulin is the next step in the signal transduction pathway, because a calmodulin antagonist, W7, reduced the effect of veratridine. A selective inhibitor of calmodulin kinases (KN‐62) and cyclosporin, an inhibitor of calcineurin, also antagonized the depolarization‐induced increase in DOR mRNA levels, which indicates that both calcium/calmodulin‐dependent enzymes are involved in the activity‐dependent induction of DOR gene expression. Induction of DOR gene expression by an activity‐dependent increase in intracellular Ca2+ concentration may serve as a feedback regulatory mechanism because activation of DOR leads to hyperpolarization and lower excitability of neurons.
Programmed Cell Death in Cortical Chick Embryo Astrocytes Is Associated with Activation of Protein Kinase PK60 and Ceramide Formation
Authors: Mangoura Dimitra , Dawson Glyn
Published: July 2010

Abstract: Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3‐hydroxy DNA ends and production of 200‐bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60‐kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose‐dependent increase in [3H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin β1 but were increased (in a dose‐dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2‐ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.
Phospholipase Cγ1 in Bovine Rod Outer Segments: Immunolocalization and Light‐Dependent Binding to Membranes
Authors: Ghalayini Abboud J. , Weber Nathan R. , Rundle Dana R. , Koutz Cynthia A. , Lambert David , Guo Xiao X. , Anderson Robert E.
Published: July 2010

Abstract: We have investigated the isozymes of a phosphoinositide‐specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ1, γ1, and δ1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light‐adapted in vitro had more PLCγ1 on immunoblots than ROS from dark‐adapted retinas. PLC enzyme activity in ROS from light‐adapted retinas was 69 and 46% higher than ROS from dark‐adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 µM. These results demonstrate the presence of PLCγ1 in bovine ROS and show that ROS prepared from light‐adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.
Pathologic Amyloid β‐Protein Cell Surface Fibril Assembly on Cultured Human Cerebrovascular Smooth Muscle Cells
Authors: Van Nostrand William E. , Melchor Jerry P. , Ruffini Lynda
Published: July 2010

Abstract: Cerebrovascular amyloid β‐protein (Aβ) deposition is a key pathological feature of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis‐Dutch type (HCHWA‐D). Aβ1–40 containing the E22Q HCHWA‐D mutation, but not wild‐type Aβ1–40, potently induces several pathologic responses in cultured human cerebrovascular smooth muscle cells, including cellular degeneration and a robust increase in the levels of cellular Aβ precursor. In the present study, we show by several quantitative criteria, including thioflavin T fluorescence binding, circular dichroism spectroscopy, and transmission electron microscopic analysis, that at a concentration of 25 µM neither HCHWA‐D Aβ1–40 nor wild‐type Aβ1–40 appreciably assembles into β‐pleated sheet‐containing fibrils in solution over a 6‐day incubation period. In contrast, at the same concentrations, HCHWA‐D Aβ1–40, but not wild‐type Aβ1–40, selectively binds and assembles into abundant fibrils on the surfaces of cultured human cerebrovascular smooth muscle cells. The simultaneous addition of an equimolar concentration of the dye Congo red prevents the cell surface fibril assembly of HCHWA‐D Aβ1–40. Moreover, Congo red effectively blocks the key pathologic responses induced by HCHWA‐D Aβ1–40 in these cells. The present findings suggest that the surface of human cerebrovascular smooth muscle cells may selectively orchestrate the assembly of pathogenic Aβ fibrils and that cell surface Aβ fibril formation plays an important role in causing the pathologic responses in these cells.
Cellular Cholesterol Storage in the Niemann‐Pick Disease Type C Mouse Is Associated with Increased Expression and Defective Processing of Apolipoprotein D
Authors: Suresh Sundar , Yan Zhijie , Patel Ramesh C. , Patel Yogesh C. , Patel Shutish C.
Published: July 2010

Abstract: Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25‐hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann‐Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up‐regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30‐fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25‐hydroxycholesterol, markedly stimulated apoD release (by five‐ to 10‐fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.
Time‐ and Region‐Specific Variations in Somatostatin Release Following Amygdala Kindling in the Rat
Authors: Simonato Michele , Bregola Gianni , Beani Lorenzo , Vezzani Annamaria , Sala Roberta , Raiteri Maurizio , Bonanno Giambattista
Published: July 2010

Abstract: Somatostatin biosynthesis is activated during and following kindling epileptogenesis. The aim of this study was to investigate whether this phenomenon translates into enhanced release of the peptide and whether it is involved in kindling maintenance. A marked increase in somatostatin‐like immunoreactivity (somatostatin‐LI) was observed in hilar interneurons of the hippocampus and in their presumed projections to the outer molecular layer 1 week, but not 1 month, after the last kindled seizure. No overt changes were observed in the striatum or in the cortex. Compared with sham‐stimulated controls, (a) in the hippocampus, high‐K+‐evoked somatostatin‐LI release was unchanged in synaptosomes taken from rats killed 7 days after the last kindled seizure but was bilaterally reduced after 30 days; (b) in the striatum, it was increased (mainly ipsilaterally to stimulation) 7, but not 30, days after the last seizure; and (c) in the cortex, somatostatin‐LI release was bilaterally increased in synaptosomes taken from kindled rats 30, but not 7, days after the last seizure. This study shows that distinct changes occur in synaptosomal somatostatin‐LI release after kindling acquisition, depending on the brain area analyzed and on the time elapsed from the last generalized seizure.
Increased Protein Oxidation in Human Substantia Nigra Pars Compacta in Comparison with Basal Ganglia and Prefrontal Cortex Measured with an Improved Dinitrophenylhydrazine Assay
Authors: Floor Erik , Wetzel Mary G.
Published: July 2010

Abstract: The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4‐dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size‐dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.
Quinolinic Acid In Vivo Synthesis Rates, Extracellular Concentrations, and Intercompartmental Distributions in Normal and Immune‐Activated Brain as Determined by Multiple‐Isotope Microdialysis
Authors: Beagles Karen E. , Morrison Paul F. , Heyes Melvyn P.
Published: July 2010

Abstract: Quinolinic acid (QUIN) kills neurons by activation of NMDA receptors that are accessed via the extracellular fluid (ECF). In vivo microdialysis was employed to quantify the dynamics of ECF QUIN levels. [13C7]QUIN was perfused through the probe for in vivo calibration to accurately quantify ECF QUIN concentrations. Osmotic pumps infused [2H3]QUIN subcutaneously to quantify blood contributions to ECF and tissue levels. Local QUIN production rates and influx and efflux rates across the blood‐brain barrier were calculated from the extraction fraction of [13C7]QUIN, probe geometry, tissue diffusion coefficients, the extracellular volume fraction, and [2H3]QUIN/QUIN ratios in blood and dialysates. In normal brain, 85% of ECF QUIN levels (110 nM) originated from blood, whereas 59% of tissue homogenate QUIN (130 pmol/g) originated from local de novo synthesis. During systemic immune activation (intraperitoneal injection of endotoxin), blood QUIN levels increased (10.2‐fold) and caused a rise in homogenate (10.8‐fold) and ECF (18.5‐fold) QUIN levels with an increase in the proportions of QUIN derived from blood. During CNS inflammation (local infusion of endotoxin), increases in brain homogenate (246‐fold) and ECF (66‐fold) QUIN levels occurred because of an increase in local synthesis rate (146‐fold) and a reduction in efflux/influx ratio (by 53%). These results demonstrate that brain homogenate measures are a reflection of ECF concentrations, although there are quantitative differences in the values obtained. The mechanisms that maintain ECF QUIN levels at low values cannot do so when there are large increases in local brain synthesis or when there are large elevations in blood QUIN concentrations.
Composition and Biophysical Properties of Myelin Lipid Define the Neurological Defects in Galactocerebroside‐ and Sulfatide‐Deficient Mice
Authors: Bosio Andreas , Binczek Erika , Haupt Walter F. , Stoffel Wilhelm
Published: July 2010

Abstract: Oligodendrocytes and Schwann cell‐specific proteins are assembled with a highly ordered membrane lipid bilayer to the myelin sheath of axons, which functions as an insulator and allows rapid saltatory conduction. We approached the question of the function of the CNS and PNS myelin‐specific galactospingolipids cerebrosides and sulfatides by generating a ceramide galactosyltransferase null allelic mouse line (cgt−/−). Galactocerebroside‐ and sulfatide‐deficient myelin loses its insulating properties and causes a severe dysmyelinosis that is incompatible with life. Here, we describe the biochemical and biophysical analysis of the myelin lipid bilayer of cgt−/− mice. The lipid composition of CNS and PNS myelin of cgt−/− mice is seriously perturbed and the sphingolipid biosynthetic pathway altered. Nonhydroxy and hydroxy fatty acid‐substituted glycosylceramides (GlcC) are synthesized by oligodendrocytes and sulfated GlcC in addition in Schwann cells. The monogalactosyldiglyceride fraction is missing in the cgt−/− mouse. This new lipid composition can be correlated with the biophysical properties of the myelin sheath. The deficiency of galactocerebrosides and sulfatides leads to an increased fluidity, permeability, and impaired packing of the myelin lipid bilayer of the internodal membrane system. The loss of the two glycosphingolipid classes causes the breakdown of saltatory conductance of myelinated axons in the cgt−/− mouse.
Production of Reactive Oxygen Species and Release of l‐Glutamate During Superoxide Anion‐Induced Cell Death of Cerebellar Granule Neurons
Authors: Satoh Takumi , Numakawa Tadahiro , Abiru Yasuhiro , Yamagata Tomoko , Ishikawa Yasuyuki , Enokido Yasushi , Hatanaka Hiroshi
Published: July 2010

Abstract: Enhanced production of superoxide anion (O2−) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O2− generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C‐DCDHF‐DA) for O2− and other ROS and hydroethidine (HEt) specifically for O2− by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO‐induced cell death. Catalase effectively reduced C‐DCDHF‐DA but not HEt fluorescence, whereas SOD reduced HEt but not C‐DCDHF‐DA fluorescence, indicating that HEt and C‐DCDHF‐DA fluorescence correlated with O2− and hydrogen peroxide, respectively. The NMDA antagonist MK‐801 prevented the death. XA + XO induced an increase in l‐glutamate release from cerebellar granule neurons. These results indicate that elevation of O2− induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l‐glutamate.
Selective Acceleration of Arachidonic Acid Reincorporation into Brain Membrane Phospholipid Following Transient Ischemia in Awake Gerbil
Authors: Rabin Olivier , Chang Michael C. J. , Grange Eric , Bell Jane , Rapoport Stanley I. , Deutsch Joseph , Purdon A. David
Published: July 2010

Abstract: Awake gerbils were subjected to 5 min of forebrain ischemia by clamping the carotid arteries for 5 min and then allowing recirculation. Radiolabeled arachidonic or palmitic acid was infused intravenously for 5 min at the start of recirculation, after which the brains were prepared for quantitative autoradiography or chemical analysis. Dilution of specific activity of the acyl‐CoA pool was independently determined for these fatty acids in control gerbils and following 5 min of ischemia and 5 min of reperfusion. Using a quantitative method for measuring regional in vivo fatty acid incorporation into and turnover within brain phospholipids and determining unlabeled concentrations of acyl‐CoAs following recirculation, it was shown that reperfusion after 5 min of ischemia was accompanied by a threefold increase compared with the control in the rate of reincorporation of unlabeled arachidonate that had been released during ischemia, whereas reincorporation of released palmitate was not different from the control. Selective and accelerated reincorporation of arachidonate into brain phospholipids shortly after ischemia may ameliorate specific deleterious effects of arachidonate and its metabolites on brain membranes.
Presynaptic Modulation of Cholecystokinin Release by Protein Kinase C in the Rat Hippocampus
Authors: Breukel Alexandra I. M. , Wiegant Victor M. , Lopes da Silva Fernando H. , Ghijsen Wim E. J. M.
Published: July 2010

Abstract: The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK‐8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC‐activating phorbol ester 4β‐phorbol 12,13‐dibutyrate (β‐PDBu) dose dependently (5–5,000 nM) increased CCK‐8 release in a strictly Ca2+‐dependent way. This effect was observed only when synaptosomes were stimulated with the K+A channel blocker 4‐aminopyridine (4‐AP; 1 mM) but not with KCI (10–30 mM). The PDBu‐induced exocytosis of CCK‐8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin‐C and was not mimicked by α‐PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK‐8 exocytosis. β‐PDBu (50–100 nM) also stimulated the 4‐AP‐evoked Ca2+‐dependent release of the classic transmitter GABA, which co‐localizes with CCK‐8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid did not stimulate, but instead inhibited, both the CCK‐8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of colocalizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.
Mutagenesis of a Single Amino Acid in the Rat μ‐Opioid Receptor Discriminates Ligand Binding
Authors: Bot George , Blake Allan D. , Li Shuixing , Reisine Terry
Published: July 2010

Abstract: To investigate the role of Asp114 in the cloned rat μ‐opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant μ receptor D114N. The wild‐type μ receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The μ‐selective agonists [d‐Ala2,MePhe4,Gly‐ol5]enkephalin and morphine and the endogenous peptides Met‐enkephalin and β‐endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild‐type μ receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild‐type and D114N mutant μ receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N μ receptors to inhibit adenylyl cyclase. Although the Asp114 to Asn114 mutation reduced full‐agonist binding, mutation of His297 to Asn297 in the μ receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the μ receptor than do the prototypical full agonists.
Continuous Monitoring of Extracellular Glucose Concentrations in the Striatum of Freely Moving Rats with an Implanted Glucose Biosensor
Authors: Lowry John P. , O'Neill Robert D. , Boutelle Martyn G. , Fillenz Marianne
Published: July 2010

Abstract: We have used a glucose oxidase‐based sensor implanted in the striatum of freely moving rats to determine the concentration of extracellular glucose in two distinct ways. With a modification of the zero net flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, we calculated the concentration at which there was no change in glucose current by regression analysis; this gave a concentration of 0.351 ± 0.016 mM. Calculating the concentration from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. Infusion of 50 µM veratridine through the adjacent dialysis probe caused a steep decrease in glucose current as soon as the drug reached the brain in contrast to the delayed fall (7.5 min) seen with microdialysis in previous experiments from this laboratory. These results demonstrate that this biosensor provides a direct, real‐time measure of the extracellular concentration of glucose.
Myelin Basic Protein‐Related Proteins in Mouse Brain and Immune Tissues
Authors: Kalwy Stephan , Marty Marie‐Chantal , Bausero Pedro , Pessac Bernard
Published: July 2010

Abstract: Brain and immune system tissues express myelin basic protein (MBP) mRNAs that contain novel exons upstream of those of the classic MBPs. We have generated antisera against a recombinant protein that includes the deduced sequence for one of the predicted species of the new MBP‐related protein, hemopoietic HMBPR1. We report here the presence of multiple proteins reactive to the antisera in a range of tissues. The principal finding is that the antisera recognize a family of proteins of ∼25 kDa that are restricted to brain, thymus, and spleen, the only tissues in which the new MBP‐related transcripts are present. These antisera also detect other proteins of apparent molecular mass consistent with other isoforms of predicted MBP‐related proteins. The expression of MBP‐related proteins in immune system tissues may be important for self‐tolerance to CNS MBPs and the initiation of immune‐associated demyelinating diseases.
ETA and ETB Specific Ligands Synergistically Antagonize Endothelin‐1 Binding to an Atypical Endothelin Receptor in Primary Rat Astrocytes
Authors: Jensen Niels , Hasselblatt Martin , Sirén Anna‐Leena , Schilling Lothar , Schmidt Martin , Ehrenreich Hannelore
Published: July 2010

Abstract: Using a whole‐cell binding procedure with long incubations at low temperature and subsequent acid stripping, we have characterized an atypical endothelin (ET) receptor in primary rat cortical astrocyte cultures. We found the following: (a) no competition for 125I‐ET‐1 binding by the ETA antagonists BQ‐123 and LU 135252 or the ETB agonist IRL 1620; (b) weak competition by the ETB antagonist BQ‐788 and by the predominant ETB ligand ET‐3; (c) potent synergistic competition of ETA and ETB ligands in combination for 125I‐ET‐1 binding; (d) potent competition of ET‐1 with any of the radioligands used, 125I‐ET‐1, 125I‐IRL 1620, and [3H]BQ‐123; (e) lack of competition of IRL 1620 and BQ‐123 with the respective other radioligand; (f) shifting of the amount of acid‐strippable 125I‐ET‐1 binding from 20 to 80% by ETB ligands and to 4% by ETA ligands; and (g) as a control, typical ETA and ETB binding characteristics of the RAT‐1 fibroblast and the U373MG astrocytoma cell line, respectively, under our assay conditions. The unusual binding properties of astrocytic ET receptors described in this study appear to be the result of several binding sites in the receptor for different ET ligands or ligand epitopes.
R‐Deprenyl and R‐2‐Heptyl‐N‐Methylpropargylamine Prevent Apoptosis in Cerebellar Granule Neurons Induced by Cytosine Arabinoside but Not Low Extracellular Potassium
Authors: Paterson I. A. , Zhang D. , Warrington R. C. , Boulton A. A.
Published: July 2010

Abstract: R‐Deprenyl and R‐2‐heptyl‐N‐methylpropargylamine (R‐2‐HMP) are compounds that have been shown to reduce neuronal death in various in vitro and in vivo models involving apoptosis but do not always prevent apoptosis. In the present study we have examined the effects of these compounds and their S enantiomers on cytosine arabinoside (ara C)‐induced apoptosis and low K+‐induced apoptosis in cerebellar granule cells in primary culture. It was found that R‐deprenyl and R‐2‐HMP could prevent ara C‐induced apoptosis with an EC50 around 10−9M but could not prevent low K+‐induced apoptosis. S‐Deprenyl and S‐2‐HMP did not prevent apoptosis under any conditions but were found to antagonize the antiapoptotic actions of R‐deprenyl and R‐2‐HMP. Using the fluorescent mitochondrial dye chloromethyltetramethylrhodamine methyl ester it was found that there was a loss of mitochondrial function in cerebellar granule cells exposed to ara C but not low K+ medium. R‐Deprenyl and R‐2‐HMP prevented the ara C‐induced loss of mitochondrial function. It is concluded that R‐deprenyl and R‐2‐HMP prevent apoptosis of cerebellar granule cells by a mechanism that is independent of monoamine oxidase inhibition and that they act on the same site to prevent specifically apoptosis involving a loss of mitochondrial membrane potential, possibly p53‐dependent apoptosis.
Regulation of Secretion of Alzheimer Amyloid Precursor Protein by the Mitogen‐Activated Protein Kinase Cascade
Authors: Desdouits‐Magnen Juliette , Desdouits Frédéric , Takeda Shizu , Syu Li‐Jyun , Saltiel Alan R. , Buxbaum Joseph D. , Czernik Andrew J. , Nairn Angus C. , Greengard Paul
Published: July 2010

Abstract: Activation of protein kinase C (PKC) regulates the processing of Alzheimer amyloid precursor protein (APP) into its soluble form (sAPP) and amyloid β‐peptide (Aβ). However, little is known about the intermediate steps between PKC activation and modulation of APP metabolism. Using a specific inhibitor of mitogen‐activated protein (MAP) kinase kinase activation (PD 98059), as well as a dominant negative mutant of MAP kinase kinase, we show in various cell lines that stimulation of PKC by phorbol ester rapidly induces sAPP secretion through a mechanism involving activation of the MAP kinase cascade. In PC12‐M1 cells, activation of MAP kinase by nerve growth factor was associated with stimulation of sAPP release. Conversely, M1 muscarinic receptor stimulation, which is known to act in part through a PKC‐independent pathway, increased sAPP secretion mainly through a MAP kinase‐independent pathway. Aβ secretion and its regulation by PKC were not affected by PD 98059, supporting the concept of distinct secretory pathways for Aβ and sAPP formation.
Modeling Voltammetry and Microdialysis of Striatal Extracellular Dopamine: The Impact of Dopamine Uptake on Extraction and Recovery Ratios
Authors: Peters Jennifer L. , Michael Adrian C.
Published: July 2010

Abstract: Numerical modeling was used as a means to examine the relationship between the outcome of in vivo voltammetry and microdialysis experiments and dopamine concentrations in the extracellular fluid of rat striatum. In the case of microdialysis, quantitative interpretation of results demands knowledge of the in vivo values for the extraction and recovery ratios of the probes toward dopamine. Equality of the extraction and recovery ratios is a necessary condition for the direct application of the no‐net‐flux method as a quantitative technique. Recent results have suggested that the extraction and recovery ratios are not equal, and this interpretation is now supported by theory. A new relationship between extraction and recovery is proposed.
N‐Methyl‐d‐Aspartate Receptor Desensitisation Is Neuroprotective by Inhibiting Glutamate‐Induced Apoptotic‐Like Death
Authors: Wood Anita M. , Bristow David R.
Published: July 2010

Abstract: Glutamate excitotoxicity is implicated in several neurodegenerative diseases; consequently, considerable effort has been made to elucidate neuroprotective mechanisms against such toxicity. N‐Methyl‐d‐aspartate (NMDA) receptor desensitisation is one potential mechanism for controlling glutamate‐mediated neuronal cell death. Pretreatment of rat cerebellar granule cells with subtoxic concentrations of NMDA caused a marked reduction in the calcium signals generated by subsequent glutamate stimulation, and, furthermore, this receptor desensitisation was coupled to a reduction in glutamate‐induced apoptotic‐like death. These effects were reduced by either d‐2‐amino‐5‐phosphonopentanoic acid, an NMDA receptor antagonist, or cyclosporin A, an inhibitor of calcineurin. Thus, the results support a role for receptor desensitisation in protection from glutamate‐mediated apoptotic‐like neuronal cell death.
Specific Neurochemical Derangements of Brain Projecting Neurons in Apolipoprotein E‐Deficient Mice
Authors: Chapman Shira , Michaelson Daniel M.
Published: July 2010

Abstract: Apolipoprotein E (apoE)‐deficient mice provide a useful system for studying the role of apoE in neuronal maintenance and repair. Previous studies revealed specific memory impairments in these mice that are associated with presynaptic derangements in projecting forebrain cholinergic neurons. In the present study we examined whether dopaminergic, noradrenergic, and serotonergic projecting pathways of apoE‐deficient mice are also affected and investigated the mechanisms that render them susceptible. The densities of nerve terminals of forebrain cholinergic projections were monitored histochemically by measurements of acetylcholinesterase activity, whereas those of the dopaminergic nigrostriatal pathway, the noradrenergic locus coeruleus cortical projection, and the raphe‐cortical serotonergic tract were measured autoradiographically using radioligands that bind specifically to the respective presynaptic transporters of these neuronal tracts. The results obtained revealed that synaptic densities of cholinergic, noradrenergic, and serotonergic projections in specific brain regions of apoE‐deficient mice are markedly lower than those of controls. Furthermore, the extent of presynaptic derangement within each of these tracts was found to be more pronounced the further away the nerve terminal is from its cell body. In contrast, the nerve terminal density of the dopaminergic neurons that project from the substantia nigra to the striatum was unaffected and was similar to that of the controls. The rank order of these presynaptic derangements at comparable distances from the respective cell bodies was found to be septohippocampal cholinergic > nucleus basalis cholinergic > locus coeruleus adrenergic > raphe serotonergic ≫ nigrostriatal dopaminergic, which interestingly is similar to that observed in Alzheimer's disease. These results suggest that two complementary factors determine the susceptibility of brain projecting neurons to apoE deficiency: pathway‐specific differences and the distance of the nerve terminals from their cell body.
Selective Increases in Phosphoinositide Signaling Activity and G Protein Levels in Postmortem Brain from Subjects with Schizophrenia or Alcohol Dependence
Authors: Jope Richard S. , Song Ling , Grimes Carol A. , Pacheco Mary A. , Dilley Ginny E. , Li Xiaohua , Meltzer Herbert Y. , Overholser James C. , Stockmeier Craig A.
Published: July 2010

Abstract: Comparisons of the activity of the G protein‐mediated phosphoinositide signal transduction system and of G protein levels were made in two regions of frontal cortex from eight schizophrenic, alcohol‐dependent, and control subjects. G protein‐mediated phosphoinositide hydrolysis was measured by stimulating cortical membranes incubated with [3H]phosphatidylinositol with 0.3–10 µM guanosine 5′‐O‐(3‐thio)triphosphate (GTPγS). In frontal cortex areas 8/9, GTPγS‐induced phosphoinositide hydrolysis was 50% greater in schizophrenic than control or alcohol‐dependent subjects, whereas there were no differences among these groups of subjects in the response to GTPγS in frontal cortex area 10. Agonists for dopaminergic, cholinergic, purinergic, serotonergic, histaminergic, and glutamatergic receptors coupled to the phosphoinositide signaling system increased [3H]phosphatidylinositol hydrolysis in a GTPγS‐dependent manner. Responses to most agonists were similar in all three subject groups in both cortical regions, with the largest difference being a 40% greater response to dopaminergic receptor stimulation in frontal cortex 8/9 from schizophrenic subjects. Measurements of the levels of phospholipase C‐β, and of α‐subunits of Gq, Go, Gi1, Gi2, and Gs, made by immunoblot analyses revealed no differences among the groups of subjects except for increased Gαo in schizophrenic subjects and increased Gαo and Gαi1 in alcohol‐dependent subjects. These results demonstrate that schizophrenia is associated with increased activity of the phosphoinositide signal transduction system and increased levels of Gαo, whereas the phosphoinositide system was unaltered in alcohol dependence, but Gαo and Gαi1 were increased.