bluuebunny/crossref_metadata_2025 huggingface dataset:
Docosahexaenoic acid provides protection from impairment of learning ability in Alzheimer's disease model rats
Authors: Hashimoto Michio , Hossain Shahdat , Shimada Toshio , Sugioka Kozo , Yamasaki Hiroshi , Fujii Yoshimi , Ishibashi Yutaka , Oka Jun‐Ichiro , Shido Osamu
Published: March 2003

Docosahexaenoic acid (C22:6, n‐3), a major n‐3 fatty acid of the brain, has been implicated in restoration and enhancement of memory‐related functions. Because Alzheimer's disease impairs memory, and infusion of amyloid‐β (Aβ) peptide (1–40) into the rat cerebral ventricle reduces learning ability, we investigated the effect of dietary pre‐administration of docosahexaenoic acid on avoidance learning ability in Aβ peptide‐produced Alzheimer's disease model rats. After a mini‐osmotic pump filled with Aβ peptide or vehicle was implanted in docosahexaenoic acid‐fed and control rats, they were subjected to an active avoidance task in a shuttle avoidance system apparatus. Pre‐administration of docosahexaenoic acid had a profoundly beneficial effect on the decline in avoidance learning ability in the Alzheimer's disease model rats, associated with an increase in the cortico‐hippocampal docosahexaenoic acid/arachidonic acid molar ratio, and a decrease in neuronal apoptotic products. Docosahexaenoic acid pre‐administration furthermore increased cortico‐hippocampal reduced glutathione levels and glutathione reductase activity, and suppressed the increase in lipid peroxide and reactive oxygen species levels in the cerebral cortex and hippocampus of the Alzheimer's disease model rats, suggesting an increase in antioxidative defence. Docosahexaenoic acid is thus a possible prophylactic means for preventing the learning deficiencies of Alzheimer's disease.
Phospholipase C‐mediated signalling is not required for histamine‐induced catecholamine secretion from bovine chromaffin cells
Authors: Donald Amanda N. , Wallace Damian J. , McKenzie Sacha , Marley Philip D.
Published: March 2003

A possible role for signalling through phospholipase C in histamine‐induced catecholamine secretion from bovine adrenal chromaffin cells has been investigated. Secretion evoked by histamine over 10 min was not prevented by inhibiting inositol‐1,4,5‐trisphosphate receptors with 2‐APB, by blocking ryanodine receptors with a combination of ryanodine and caffeine, or by depleting intracellular Ca2+ stores by pretreatment with thapsigargin. Inhibition of protein kinase C with Ro31‐8220 also failed to reduce secretion. Inhibition of phospholipase C with ET‐18‐OCH3 reduced both histamine‐ and K+‐induced inositol phosphate responses by 70–80% without reducing their secretory responses. Stimulating phospholipase C with Pasteurella multocida toxin did not evoke secretion or enhance the secretory response to histamine. The secretory response to histamine was little affected by tetrodotoxin or by substituting extracellular Na+ with N‐methyl‐d‐glucamine+ or choline+, or by substituting external Cl– with nitrate–. Blocking various K+ channels with apamin, charybdotoxin, Ba2+, tetraethylammonium, 4‐aminopyridine, tertiapin or glibenclamide failed to reduce the ability of histamine to evoke secretion. These results indicate that histamine evokes secretion by a mechanism that does not require inositol‐1,4,5‐trisphosphate‐mediated mobilization of stored Ca2+, diacylglycerol‐mediated activation of protein kinase C, or activation of phospholipase C. The results are consistent with histamine acting by depolarizing chromaffin cells through a phospholipase C‐independent mechanism.
Microglial activation‐mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson's disease
Authors: Gao Hui‐Ming , Jiang Janwei , Wilson Belinda , Zhang Wanqin , Hong Jau‐Shyong , Liu Bin
Published: March 2003

The etiology of sporadic Parkinson's disease (PD) remains unknown. Increasing evidence has suggested a role for inflammation in the brain in the pathogenesis of PD. However, it has not been clearly demonstrated whether microglial activation, the most integral part of the brain inflammatory process, will result in a delayed and progressive degeneration of dopaminergic neurons in substantia nigra, a hallmark of PD. We report here that chronic infusion of an inflammagen lipopolysaccharide at 5 ng/h for 2 weeks into rat brain triggered a rapid activation of microglia that reached a plateau in 2 weeks, followed by a delayed and gradual loss of nigral dopaminergic neurons that began at between 4 and 6 weeks and reached 70% by 10 weeks. Further investigation of the underlying mechanism of action of microglia‐mediated neurotoxicity using rat mesencephalic neuron‐glia cultures demonstrated that low concentrations of lipopolysaccharide (0.1–10 ng/mL)‐induced microglial activation and production of neurotoxic factors preceded the progressive and selective degeneration of dopaminergic neurons. Among the factors produced by activated microglia, the NADPH oxidase‐mediated release of superoxide appeared to be a predominant effector of neurodegeneration, consistent with the notion that dopaminergic neurons are particularly vulnerable to oxidative insults. This is the first report that microglial activation induced by chronic exposure to inflammagen was capable of inducing a delayed and selective degeneration of nigral dopaminergic neurons and that microglia‐originated free radicals play a pivotal role in dopaminergic neurotoxicity in this inflammation‐mediated model of PD.
Decreased TCA cycle rate in the rat brain after acute 3‐NP treatment measured by in vivo1H‐{13C} NMR spectroscopy
Authors: Henry Pierre‐Gilles , Lebon Vincent , Vaufrey Françoise , Brouillet Emmanuel , Hantraye Philippe , Bloch Gilles
Published: March 2003

Inhibition of succinate dehydrogenase (SDH) by the mitochondrial toxin 3‐nitropropionic acid (3‐NP) has gained acceptance as an animal model of Huntington's disease. In this study 13C NMR spectroscopy was used to measure the tricarboxylic acid (TCA) cycle rate in the rat brain after 3‐NP treatment. The time course of both glutamate C4 and C3 13C labelling was monitored in vivo during an infusion of [1‐13C]glucose. Data were fitted by a mathematical model to yield the TCA cycle rate (Vtca) and the exchange rate between α‐ketoglutarate and glutamate (Vx). 3‐NP treatment induced a 18% decrease in Vtca from 0.71 ± 0.02 µmol/g/min in the control group to 0.58 ± 0.02 µmol/g/min in the 3‐NP group (p < 0.001). Vx increased from 0.88 ± 0.08 µmol/g/min in the control group to 1.33 ± 0.24 µmol/g/min in the 3‐NP group (p < 0.07). Fitting the C4 glutamate time course alone under the assumption that Vx is much higher than Vtca yielded Vtca=0.43 µmol/g/min in both groups. These results suggest that both Vtca and Vx are altered during 3‐NP treatment, and that both glutamate C4 and C3 labelling time courses are necessary to obtain a reliable measurement of Vtca.
Region‐selective effects of neuroinflammation and antioxidant treatment on peripheral benzodiazepine receptors and NMDA receptors in the rat brain
Authors: Biegon A. , Alvarado M. , Budinger T. F. , Grossman R. , Hensley K. , West M. S. , Kotake Y. , Ono M. , Floyd R. A.
Published: March 2003

Following induction of acute neuroinflammation by intracisternal injection of endotoxin (lipopolysaccharide) in rats, quantitative autoradiography was used to assess the regional level of microglial activation and glutamate (NMDA) receptor binding. The possible protective action of the antioxidant phenyl‐tert‐butyl nitrone in this model was tested by administering the drug in the drinking water for 6 days starting 24 hafter endotoxin injection. Animals were killed 7 days post‐injection and consecutive cryostat brain sections labeled with [3H]PK11195 as a marker of activated microglia and [125I]iodoMK801 as a marker of the open‐channel, activated state of NMDA receptors. Lipopolysaccharide increased [3H]PK11195 binding in the brain, with the largest increases (two‐ to threefold) in temporal and entorhinal cortex, hippocampus, and substantia innominata. A significant (> 50%) decrease in [125I]iodoMK801 binding was found in the same brain regions. Phenyl‐tert‐butyl nitrone treatment resulted in a partial inhibition (approx. 25% decrease) of the lipopolysaccharide‐induced increase in [3H]PK11195 binding but completely reversed the lipopolysaccharide‐induced decrease in [125I]iodoMK80 binding in the entorhinal cortex, hippocampus, and substantia innominata. Loss of NMDA receptor function in cortical and hippocampal regions may contribute to the cognitive deficits observed in diseases with a neuroinflammatory component, such as meningitis or Alzheimer's disease.
Endoplasmic reticulum calcium release is modulated by actin polymerization
Authors: Wang Yue , Mattson Mark P. , Furukawa Katsutoshi
Published: March 2003

Intracellular calcium ions regulate the structure and functions of cytoskeletal proteins. On the other hand, recent studies have shown that the cytoskeleton, and actin filaments in particular, can modulate calcium influx through plasma membrane ligand‐ and voltage‐gated channels. We now report that calcium release from inositol trisphosphate (IP3) and ryanodine‐sensitive endoplasmic reticulum (ER) stores is modulated by polymerization and depolymerization of actin filaments in cultured hippocampal neurons. Depolymerization of actin filaments with cytochalasin D attenuates calcium release induced by carbamylcholine (CCh; a muscarinic agonist for IP3 pathway), caffeine (a ryanodine receptor agonist) and thapsigargin (an inhibitor of the ER calcium‐ ATPase) in both the presence and absence of extracellular calcium. Conversely, the actin polymerizing agent jasplakinolide potentiates calcium release induced by CCh, caffeine and thapsigargin. Cytochalasin D attenuated, while jasplakinolide augmented, thapsigargin‐induced JNK activation and neuronal cell death. Our data show that the actin cytoskeleton regulates ER calcium release, suggesting roles for actin in the various physiological and pathological processes that involve calcium release.
Regional and cellular expression of CYP2D6 in human brain: higher levels in alcoholics
Authors: Miksys Sharon , Rao Yushu , Hoffmann Ewa , Mash Deborah C. , Tyndale Rachel F.
Published: March 2003

Cytochrome P450 (CYP) 2D6 is expressed in liver, brain and other extrahepatic tissues where it metabolizes a range of centrally acting drugs and toxins. As ethanol can induce CYP2D in rat brain, we hypothesized that CYP2D6 expression is higher in brains of human alcoholics. We examined regional and cellular expression of CYP2D6 mRNA and protein by RT‐PCR, Southern blotting, slot blotting, immunoblotting and immunocytochemistry. A significant correlation was found between mean mRNA and CYP2D6 protein levels across 13 brain regions. Higher expression was detected in 13 brain regions of alcoholics (n = 8) compared to nonalcoholics (n = 5) (anovap < 0.0001). In hippocampus this was localized in CA1–3 pyramidal cells and dentate gyrus granular neurons. In cerebellum this was localized in Purkinje cells and their dendrites. Both of these brain regions, and these same cell‐types, are known to be susceptible to alcohol damage. For one case, a poor metabolizer (CYP2D6*4/*4), there was no detectable CYP2D6 protein, confirming the specificity of the antibody used. These data suggest that in alcoholics elevated brain CYP2D6 expression may contribute to altered sensitivity to centrally acting drugs and to the mediation of neurotoxic and behavioral effects of alcohol.
Evidence that brain‐derived neurotrophic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice
Authors: Lee Jaewon , Duan Wenzhen , Mattson Mark P.
Published: March 2003

To determine the role of brain‐derived neurotrophic factor (BDNF) in the enhancement of hippocampal neurogenesis resulting from dietary restriction (DR), heterozygous BDNF knockout (BDNF +/–) mice and wild‐type mice were maintained for 3 months on DR or ad libitum (AL) diets. Mice were then injected with bromodeoxyuridine (BrdU) and killed either 1 day or 4 weeks later. Levels of BDNF protein in neurons throughout the hippocampus were decreased in BDNF +/– mice, but were increased by DR in wild‐type mice and to a lesser amount in BDNF +/– mice. One day after BrdU injection the number of BrdU‐labeled cells in the dentate gyrus of the hippocampus was significantly decreased in BDNF +/– mice maintained on the AL diet, suggesting that BDNF signaling is important for proliferation of neural stem cells. DR had no effect on the proliferation of neural stem cells in wild‐type or BDNF +/– mice. Four weeks after BrdU injection, numbers of surviving labeled cells were decreased in BDNF +/– mice maintained on either AL or DR diets. DR significantly improved survival of newly generated cells in wild‐type mice, and also improved their survival in BDNF +/– mice, albeit to a lesser extent. The majority of BrdU‐labeled cells in the dentate gyrus exhibited a neuronal phenotype at the 4‐week time point. The reduced neurogenesis in BDNF +/– mice was associated with a significant reduction in the volume of the dentate gyrus. These findings suggest that BDNF plays an important role in the regulation of the basal level of neurogenesis in dentate gyrus of adult mice, and that by promoting the survival of newly generated neurons BDNF contributes to the enhancement of neurogenesis induced by DR.
Expression of gp130 and leukaemia inhibitory factor receptor subunits in adult rat sensory neurones: regulation by nerve injury
Authors: Gardiner Natalie J. , Cafferty William B. J. , Slack Sarah E. , Thompson Stephen W. N.
Published: March 2003

Members of the interleukin‐6 (IL‐6) family of cytokines have been implicated as major mediators of the response of the adult nervous system to injury. The basis for an interaction of IL‐6 cytokines with adult sensory neurones has been established by analysing the levels and distribution of the two signal‐transducing receptor subunits, glycoprotein 130 (gp130) and leukaemia inhibitory factor receptor (LIFR), in the dorsal root ganglion (DRG) of male adult rats before and following nerve injury. All sensory neurones express gp130‐immunoreactivity (IR) in the cytoplasm and on the plasma membrane. Levels of gp130 and its intracellular distribution remained unchanged up to 14 days following sciatic nerve axotomy. LIFR‐IR was largely absent from the cytoplasm and plasma membrane of sensory neurones, but confined almost exclusively to the nuclear compartment. However, following axotomy, punctate cytoplasmic LIFR‐IR was detected which persisted up to 28 days following axotomy. The expression of cytoplasmic LIFR 2 days post‐axotomy was proportionally greater in a subset of small diameter sensory neurones which expressed either the sensory neuropeptide CGRP or the cell surface marker isolectin B4. The coexpression of gp130 and LIFR in the same intracellular compartment following axotomy conveys potential responsiveness of injured sensory neurones to members of the IL‐6 family of cytokines.
cDNA array reveals differential gene expression following chronic neuroleptic administration: implications of synapsin II in haloperidol treatment
Authors: Chong Victor Z. , Young L. Trevor , Mishra Ram K.
Published: March 2003

The cDNA expression array is a recently developed scientific tool that can profile the differential expression of several hundreds of genes simultaneously and is therefore advantageous in the study of antipsychotic drug action at the genetic level. Using this technology, we discovered 14 genes in the rat striatum whose expression was changed by ≥ 50% following chronic haloperidol treatment. Among them was the synapsin II gene, which was found to be significantly up‐regulated after the treatment. Since recent studies have implicated this gene in schizophrenia, further experiments were performed to determine whether chronic haloperidol exposure resulted in concurrent increases in the expression of striatal synapsin II protein. Immunoblotting revealed that protein levels of both the a and b isoforms of synapsin II were also increased by comparable amounts following haloperidol treatment. This study is the first to show the regulation of synapsin II expression by haloperidol at the transcript and protein level in rat striatum. A possible mechanism for the observed haloperidol‐induced increase in striatal synapsin II expression, along with the implications of this up‐regulation in chronic haloperidol treatment, is presented.
Mitochondrial electron transport chain complex dysfunction in a transgenic mouse model for amyotrophic lateral sclerosis
Authors: Jung Cheolwha , Higgins Cynthia M. J. , Xu Zuoshang
Published: March 2003

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease that causes degeneration of motoneurons. Mutation of Cu,Zn superoxide dismutase (SOD1) is one cause for this disease. In mice, expression of mutant protein causes motoneuron degeneration and paralysis resembling the human disease. Morphological change, indicative of mitochondrial damage, occurs at early stages of the disease. To determine whether mitochondrial function changes during the course of disease progression, enzyme activities of mitochondrial electron transport chain in spinal cords from mice at different disease stages were measured using three different methods: spectrophotometric assay, in situ histochemical enzyme assay, and blue native gel electrophoresis combined with in‐gel histochemical reaction. The enzyme activities were decreased in the spinal cord, particularly in the ventral horn, beginning at early disease stages. This decrease persisted throughout the course of disease progression. This decrease was not detected in the spinal cords of non‐transgenic animals, of mice expressing the wild‐type protein, and in cerebellum and dorsal horn of the spinal cords from mice expressing mutant protein. These results demonstrate a functional defect in mitochondria in the ventral horn region and support the view that mitochondrial damage plays a role in mutant SOD1‐induced motoneuron degeneration pathway.
Internalization of basic fibroblast growth factor at the mouse blood–brain barrier involves perlecan, a heparan sulfate proteoglycan
Authors: Deguchi Yoshiharu , Okutsu Hiroshi , Okura Takashi , Yamada Shizuo , Kimura Ryohei , Yuge Takuro , Furukawa Akihiko , Morimoto Kazuhiro , Tachikawa Masanori , Ohtsuki Sumio , Hosoya Ken‐ichi , Terasaki Tetsuya
Published: March 2003

In this study, the internalization mechanism of basic fibroblast growth factor (bFGF) at the blood–brain barrier (BBB) was investigated using a conditionally immortalized mouse brain capillary endothelial cell line (TM‐BBB4 cells) as an in vitro model of the BBB and the corresponding receptor was identified using immunohistochemical analysis. The heparin‐resistant binding of [125I]bFGF to TM‐BBB4 cells was found to be time‐, temperature‐, osmolarity‐ and concentration‐dependent. Kinetic analysis of the cell‐surface binding of [125I]bFGF to TM‐BBB4 cells revealed saturable binding with a half‐saturation constant of 76 ± 24 nm and a maximal binding capacity of 183 ± 17 pmol/mg protein. The heparin‐resistant binding of [125I]bFGF to TM‐BBB4 was significantly inhibited by a cationic polypeptide poly‐L‐lysine (300 µm), and compounds which contain a sulfate moiety, e.g. heparin and chondroitin sulfate‐B (each 10 µg/mL). Moreover, the heparin‐resistant binding of [125I]bFGF in TM‐BBB4 cells was significantly reduced by 50% following treatment with sodium chlorate, suggesting the loss of perlecan (a core protein of heparan sulfate proteoglycan, HSPG) from the extracellular matrix of the cells. This type of binding is consistent with the involvement HSPG‐mediated endocytosis. RT‐PCR analysis revealed that HSPG mRNA and FGFR1 and FGFR2 (tyrosine‐kinase receptors for bFGF) mRNA are expressed in TM‐BBB4 cells. Moreover, immunohistochemical analysis demonstrated that perlecan is expressed on the abluminal membrane of the mouse brain capillary. These results suggest that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB. HSPG at the BBB may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocytes.
Abnormal Tau phosphorylation of the Alzheimer‐type also occurs during mitosis
Authors: Delobel Patrice , Flament Stéphane , Hamdane Malika , Mailliot Christel , Sambo Anne‐Véronique , Bégard Séverine , Sergeant Nicolas , Delacourte André , Vilain Jean‐Pierre , Buée Luc
Published: March 2003

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into filaments and it may be related to the reactivation of mitotic mechanisms. In order to investigate the link between Tau phosphorylation and mitosis, Xenopus laevis oocytes in which most of the M‐phase regulators have been discovered were used as a cell model. The human Tau isoform htau412 (2+3–10+) was microinjected into prophase I oocytes that were then stimulated by progesterone that activate cyclin‐dependent kinase pathways. Hyperphosphorylation of the Tau isoform, which is characterized by a decrease of its electrophoretic mobility and its labelling by a number of phosphorylation‐dependent antibodies, was observed at the time of germinal vesicle breakdown. Surprisingly, Tau immunoreactivity, considered as typical of Alzheimer's pathology (AT100 and phospho‐Ser422), was observed in meiosis II. Because meiosis II is considered as a mitosis‐like phase, we investigated if our observation was also relevant to a neurone‐like model. Abnormal Tau phosphorylation was detected in mitotic human neuroblastoma SY5Y cells overexpressing Tau. Regarding AT100‐immunoreactivity and phospho‐Ser422, we suggest that phosphatase 2A inhibition and a phosphorylation combination of mitotic kinases may lead to this Alzheimer‐type phosphorylation. Our results not only demonstrate the involvement of mitotic kinases in Alzheimer‐type Tau phosphorylation but also indicate that Xenopus oocyte could be a useful model to identify the kinases involved in this process.
Oligodendrocyte‐myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth
Authors: Kottis Vicky , Thibault Pierre , Mikol Daniel , Xiao Zhi‐Cheng , Zhang Rulin , Dergham Pauline , Braun Peter E.
Published: March 2003

A protein fraction purified from bovine brain myelin, previously called arretin because of its ability to inhibit neurite outgrowth, has been identified as consisting predominantly of oligodendrocyte‐myelin glycoprotein (OMgp). We show that it is a potent inhibitor of neurite outgrowth from rat cerebellar granule and hippocampal cells; from dorsal root ganglion explants in which growth cone collapse was observed; from rat retinal ganglion neurons; and from NG108 and PC12 cells. OMgp purified by a different procedure from both mouse and human myelin behaves identically in all bioassays tested.
Neuronal oxidative damage from activated innate immunity is EP2 receptor‐dependent
Authors: Montine Thomas J. , Milatovic Dejan , Gupta Ramesh C. , Valyi‐Nagy Tibor , Morrow Jason D. , Breyer Richard M.
Published: March 2003

Increase in prostaglandin (PG) E2 levels and oxidative damage are associated with diseases of brain that involve activation of innate immunity. We tested the hypothesis that cerebral oxidative damage resulting from activation of innate immunity with intracerebroventricular (icv) lipopolysaccharide (LPS) is dependent on PGE2‐mediated signaling. We measured two quantitative in vivo biomarkers of lipid peroxidation: F2‐isoprostanes (IsoPs) that derive from arachidonic acid (AA) that is uniformly distributed in all cell types in brain, and F4‐neuroprostanes (NeuroPs) that derive from docosahexaenoic acid (DHA) that is highly concentrated in neuronal membranes. LPS stimulated delayed elevations in cerebral F2‐IsoPs and F4‐NeuroPs that were completely suppressed by indomethacin or ibuprofen pre‐treatment. LPS‐induced cerebral oxidative damage was abolished by disruption of subtype 2 receptor for PGE2 (EP2). In contrast, initial oxidative damage from icv kainic acid (KA) was more rapid than with LPS also was completely suppressed by indomethacin or ibuprofen pre‐treatment but was independent of EP2 receptor activation. The protective effect of deleting the EP2 receptor was not associated with changes in cerebral eicosaniod production, but was partially related to reduced induction of nitric oxide synthase (NOS) activity. These results suggest the EP2 receptor as a therapeutic target to limit oxidative damage from activation of innate immunity in cerebrum.
Polyethylene glycol immediately repairs neuronal membranes and inhibits free radical production after acute spinal cord injury
Authors: Luo Jian , Borgens Richard , Shi Riyi
Published: March 2003

Membrane disruption and the production of reactive oxygen species (ROS) are important factors causing immediate functional loss, progressive degeneration, and death in neurons and their processes after traumatic spinal cord injury. Using an in vitro guinea pig spinal cord injury model, we have shown that polyethylene glycol (PEG), a hydrophilic polymer, can significantly accelerate and enhance the membrane resealing process to restore membrane integrity following controlled compression. As a result of PEG treatment, injury‐induced ROS elevation and lipid peroxidation (LPO) levels were significantly suppressed. We further show that PEG is not an effective free radical scavenger nor does it have the ability to suppress xanthine oxidase, a key enzyme in generating superoxide. These observations suggest that it is the PEG‐mediated membrane repair that leads to ROS and LPO inhibition. Furthermore, our data also imply an important causal effect of membrane disruption in generating ROS in spinal cord injury, suggesting membrane repair to be an effective target in reducing ROS genesis.
Effect of single and repeated methamphetamine treatment on neurotransmitter release in substantia nigra and neostriatum of the rat
Authors: Bustamante Diego , You Zhi‐Bing , Castel Marie‐Noëlle , Johansson Sara , Goiny Michel , Terenius Lars , Hökfelt Tomas , Herrera‐Marschitz Mario
Published: March 2003

The main purpose of this study was to characterize the initial neurotransmission cascade elicited by methamphetamine, analysing simultaneously with in vivo microdialysis monoamine, amino acid and neuropeptide release in substantia nigra and neostriatum of the rat. The main effect of a single systemic dose of methamphetamine (15 mg/kg, subcutaneously) was an increase in dopamine levels, both in substantia nigra (≈ 10‐fold) and neostriatum (≈ 40‐fold), accompanied by a significant, but lesser, increase in dynorphin B (≈ two‐fold, in both regions), and a decrease in monoamine metabolites. A similar effect was also observed after local administration of methamphetamine (100 µm) via the microdialysis probes, but restricted to the treated region. In other experiments, rats were repeatedly treated with methamphetamine or saline, with the last dose administered 12 h before microdialysis. Dopamine K+‐stimulated release was decreased following repeated methamphetamine administration compared with that following saline, both in the substantia nigra (by ≈ 65%) and neostriatum (by ≈ 20%). In contrast, the effect of K+‐depolarization on glutamate, aspartate and GABA levels was increased following repeated administration of methamphetamine. In conclusion, apart from an impairment of monoamine neurotransmission, repeated methamphetamine produces changes in amino acid homeostasis, probably leading to NMDA‐receptor overstimulation.
Characterization of the dehydroepiandrosterone (DHEA) metabolism via oxysterol 7α‐hydroxylase and 17‐ketosteroid reductase activity in the human brain
Authors: Steckelbroeck Stephan , Watzka Matthias , Lütjohann Dieter , Makiola Paul , Nassen Alexander , Hans Volkmar H. J. , Clusmann Hans , Reissinger Annette , Ludwig Michael , Siekmann Lothar , Klingmüller Dietrich
Published: March 2003

Dehydroepiandrosterone and its sulphate are important factors for vitality, development and functions of the CNS. They were found to be subjects to a series of enzyme‐mediated conversions within the rodent CNS. In the present study, we were able to demonstrate for the first time that membrane‐associated dehydroepiandrosterone 7α‐hydroxylase activity occurs within the human brain. The cytochrome P450 enzyme demonstrated a sharp pH optimum between 7.5 and 8.0 and a mean KM value of 5.4 µm, corresponding with the presence of the oxysterol 7α‐hydroxylase CYP7B1. Real‐time RT–PCR analysis verified high levels of CYP7B1 mRNA expression in the human CNS. The additionally observed conversion of dehydroepiandrosterone via cytosolic 17β‐hydroxysteroid dehydrogenase activity could be ascribed to the activity of an enzyme with a broad pH optimum and an undetectably high KM value. Subsequent experiments with cerebral neocortex and subcortical white matter specimens revealed that 7α‐hydroxylase activity is significantly higher in the cerebral neocortex than in the subcortical white matter (p < 0.0005), whereas in the subcortical white matter, 17β‐hydroxysteroid dehydrogenase activity is significantly higher than in the cerebral neocortex (p < 0.0005). No sex differences were observed. In conclusion, the high levels of CYP7B1 mRNA in brain tissue as well as in a variety of other tissues in combination with the ubiquitous presence of 7α‐hydroxylase activity in the human temporal lobe led us to assume a neuroprotective function of the enzyme such as regulation of the immune response or counteracting the deleterious effects of neurotoxic glucocorticoids, rather than a distinct brain specific function such as neurostimulation or neuromodulation.
In depolarized and glucose‐deprived neurons, Na+ influx reverses plasmalemmal K+‐dependent and K+‐independent Na+/Ca2+ exchangers and contributes to NMDA excitotoxicity
Authors: Czyż Aneta , Kiedrowski Lech
Published: March 2003

Cerebellar granule cells (CGCs) express K+‐dependent (NCKX) and K+‐independent (NCX) plasmalemmal Na+/Ca2+ exchangers which, under plasma membrane‐depolarizing conditions and high cytosolic [Na+], may reverse and mediate potentially toxic Ca2+ influx. To examine this possibility, we inhibited NCX or NCKX with KB‐R7943 or K+‐free medium, respectively, and studied how gramicidin affects cytosolic [Ca2+] and 45Ca2+ accumulation. Gramicidin forms pores permeable to alkali cations but not Ca2+. Therefore, gramicidin‐induced Ca2+ influx is indirect; it results from fluxes of monovalent cations. In the presence of Na+, but not Li+ or Cs+, gramicidin induced Ca2+ influx that was inhibited by simultaneous application of KB‐R7943 and K+‐free medium. The data indicate that gramicidin‐induced Na+ influx reverses NCX and NCKX. To test the role of NCX and/or NCKX in excitotoxicity, we studied how NMDA affects the viability of glucose‐deprived and depolarized CGCs. To assure depolarization of the plasma membrane, we inhibited Na+,K+‐ATPase with ouabain. Although inhibition of NCX or NCKX reversal failed to significantly limit 45Ca2+ accumulation and excitotoxicity, simultaneously inhibiting NCX and NCKX reversal was neuroprotective and significantly decreased NMDA‐induced 45Ca2+ accumulation. Our data suggest that NMDA‐induced Na+ influx reverses NCX and NCKX and leads to the death of depolarized and glucose‐deprived neurons.
Constitutively and autonomously active protein kinase C associated with 14‐3‐3 ζ in the rodent brain
Authors: Dai Jian‐Guo , Murakami Kentaro
Published: March 2003

Persistent activation of protein kinase C (PKC) is required for the expression of synaptic plasticity in the brain. There are several mechanisms proposed that can lead to the prolonged activation of PKC. These include long lasting production of lipid activators (diacylglycerol and fatty acid) through mitogen‐activated protein (MAP) kinase pathway, and a modification of PKC by reactive oxygen species. In nerve growth factor (NGF)‐differentiated PC12 cells, we found that constitutive and autonomous Ca2+‐independent PKC activity is associated with 14‐3‐3 ζ. Because PKC and 14‐3‐3 ζ are both involved in synaptic plasticity and learning and memory, we examined whether PKC interacts with 14‐3‐3 ζ in the brain and whether the PKC/14‐3‐3 ζ complex has autonomous activity. Here we show that three subclasses of PKC, Ca2+‐dependent classical PKC, Ca2+‐independent novel PKC, and Ca2+‐independent and diacylglycerol‐insensitive atypical PKC, all interact with 14‐3‐3 ζ in the rodent brain. The pool size of 14‐3‐3 ζ bound form of PKC is small (1–4% of each PKC isoform), but they show constitutive and autonomous activity. Our study indicates that the binding of PKC with 14‐3‐3 ζ is at least in part independent of phosphorylation of PKC and that the C1 domain of PKC is involved in the binding. As both molecules are enriched in synaptic locus, the constitutive PKC activity and its interaction with 14‐3‐3 ζ could be a mechanism for the persistent PKC activation in the brain.
The synaptophysin/synaptobrevin interaction critically depends on the cholesterol content
Authors: Mitter Diana , Reisinger Clemens , Hinz Britta , Hollmann Susanne , Yelamanchili Sowmya V. , Treiber‐Held Stephanie , Ohm Thomas G. , Herrmann Andreas , Ahnert‐Hilger Gudrun
Published: March 2003

Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol‐binding protein. Depleting the membranous cholesterol content by filipin or β‐methylcyclodextrin (β‐MCD) decreased the solubility of synaptophysin in Triton X‐100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon β‐MCD treatment as revealed by chemical cross‐linking. Mice with a genetic mutation in the Niemann–Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo‐ or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down‐regulated after depleting the endogenous cholesterol content by the HMG‐CoA‐reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up‐regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.
Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3‐kinase in the rat hippocampus
Authors: Takagi Norio , Sasakawa Kyoko , Besshoh Shintaro , Miyake‐Takagi Keiko , Takeo Satoshi
Published: March 2003

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3‐kinase (PI3‐kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3‐kinase p85 was co‐precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3‐kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5‐fold relative to sham) was greater than in CA1 (1.7‐fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3‐kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.
Altered levels and distribution of microtubule‐associated proteins before disease onset in a mouse model of amyotrophic lateral sclerosis
Authors: Farah C. Abi , Nguyen M. D. , Julien J. P. , Leclerc N.
Published: March 2003

Alterations of the axonal transport and microtubule network are potential causes of motor neurodegeneration in mice expressing a mutant form of the superoxide dismutase 1 (SOD1G37R) linked to amyotrophic lateral sclerosis (ALS). In the present study, we investigated the biology of microtubule‐associated proteins (MAPs), responsible for the formation and stabilization of microtubules, in SOD1G37R mice. Our results show that the protein levels of MAP2, MAP1A, tau 100 kDa and tau 68 kDa species decrease significantly as early as 5 months before onset of symptoms in the spinal cord of SOD1G37R mice, whereas decrease in levels of tau 52–55 kDa species is most often noted with the manifestation of the clinical symptoms. Interestingly, there was no change in the protein levels of MAPs in the brain of SOD1G37R mice, a CNS organ spared by the mutant SOD1 toxicity. Remarkably, as early as 5 months before disease onset, the binding affinities of MAP1A, MAP2 and tau isoforms to the cytoskeleton decreased in spinal cord of SOD1G37R mice. This change correlated with a hyperphosphorylation of the soluble tau 52–55 kDa species at epitopes recognized by the antibodies AT8 and PHF‐1. Finally, a shift in the distribution of MAP2 from the cytosol to the membrane is detected in SOD1G37R mice at the same stage. Thus, alterations in the integrity of microtubules are early events of the neurodegenerative processes in SOD1G37R mice.
Martentoxin, a novel K+‐channel‐blocking peptide: purification, cDNA and genomic cloning, and electrophysiological and pharmacological characterization
Authors: Ji Yong‐Hua , Wang Wei‐Xi , Ye Jian‐Guo , He Lin‐Lin , Li Ya‐Jun , Yan Yan‐Ping , Zhou Zhuan
Published: March 2003

Martentoxin, a novel K+‐channel‐specific peptide has been purified and characterized from the venom of the East‐Asian scorpion (Buthus martensi Karsch). The whole cDNA precursor sequence suggested that martentoxin was composed of 37 residues with a unique sequence compared with other scorpion neurotoxins. The genomic DNA of martentoxin showed an additional intron situated unexpectedly in the 5′ UTR region, besides one located close to the C‐terminal of the signal peptide. The patch‐clamp recording found that martentoxin at the applied dose of 100 nm could strongly block large‐conductance Ca2+‐activated K+ (BKCa) currents in adrenal medulla chromaffin cells, and BKCa currents blocked by martentoxin could be fully recovered within 30 seconds after washing, which is at least 10 times faster than recovery after charybdotoxin. Meanwhile, a biosensor binding assay showed a fast association rate and a slow dissociation rate of martentoxin binding on rat brain synaptosomes. The binding of martentoxin on rat brain synaptosomes could be inhibited regularly by charybdotoxin, and gradually by toosendanin in a concentration‐dependent manner, but not by either apamin or P03 from Buthus martensi. The results thus indicate that martentoxin is a new member in the family of K+‐channel‐blocking ligands.
Co‐expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic‐like microvesicles in rat pinealocytes
Authors: Morimoto Riyo , Hayashi Mitsuko , Yatsushiro Shouki , Otsuka Masato , Yamamoto Akitsugu , Moriyama Yoshinori
Published: March 2003

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l‐glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l‐glutamate in synaptic‐like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l‐glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT–PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co‐localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l‐glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.
GABA‐induced neurite outgrowth of cerebellar granule cells is mediated by GABAA receptor activation, calcium influx and CaMKII and erk1/2 pathways
Authors: Borodinsky Laura N. , O'Leary Deirdre , Neale Joseph H. , Vicini Stefano , Coso Omar A. , Fiszman Mónica L.
Published: March 2003

During neuronal development, GABAA‐mediated responses are depolarizing and induce an increase in the intracellular calcium concentration. Since calcium oscillations can modulate neurite outgrowth, we explored the capability of GABA to induce changes in cerebellar granule cell morphology. We find that treatment with GABA (1–1000 µm) induces an increase in the intracellular calcium concentration through the activation of GABAA receptors and voltage‐gated calcium channels of the L‐subtype. Perforated patch‐clamp recordings reveal that this depolarizing response is due to a chloride reversal potential close to − 35 mV. When cells are grown in depolarizing potassium chloride concentrations, a shift in reversal potential (Erev) for GABA is observed, and only 20% of the cells are depolarized by the neurotransmitter at day 5 in vitro. On the contrary, cells grown under resting conditions are depolarized after GABA application even at day 8. GABA increases the complexity of the dendritic arbors of cerebellar granule neurons via a calcium‐dependent mechanism triggered by voltage‐gated calcium channel activation. Specific blockers of calcium‐calmodulin kinase II and mitogen‐activated protein kinase kinase (KN93 and PD098059) implicate these kinases in the intracellular pathways involved in the neuritogenic effect of GABA. These data demonstrate that GABA exerts a stimulatory role on cerebellar granule cell neuritogenesis through calcium influx and activation of calcium‐dependent kinases.
Organization, work and work reactions: a study of the relationship between organizational aspects of nursing and nurses' work characteristics and work reactions
Authors: Tummers Gladys E.R. , Landeweerd Jan A. , Van Merode Godefridus G.
Published: March 2003

Organization, work and work reactions: a study of the relationship between organizational aspects of nursing and nurses' work characteristics and work reactions

This article describes a quantitative, correlational study of the relationship between organizational aspects (such as the predictability of the care), work characteristics (autonomy and workload) and work reactions (work satisfaction and health complaints) in nursing work. The variables have been measured by questionnaires. Subjects were 155 nurses from nine units in two general hospitals in the Netherlands. Several organizational aspects seem to be correlated with work characteristics and work reactions. Organizational aspects particularly influence work characteristics and play, therefore, a mediating role in the relationship between organizational aspects and work reactions. Job satisfaction is not only correlated with the autonomy, but also with workload. Health complaints appeared to be mainly correlated with the workload. It is concluded that for an improvement of job satisfaction and reduction of health complaints, it is not only necessary to improve autonomy and reduce workload, but also to pay attention to organizational aspects because these may influence the work characteristics.
Dental phobic patients' view of dental anxiety and experiences in dental care: a qualitative study
Authors: Abrahamsson Kajsa H. , Berggren Ulf , Hallberg Lillemor , Carlsson Sven G.
Published: March 2003

Dental phobic patients' view of dental anxiety and experiences in dental care: a qualitative study

The aim of this study was to explore and describe dental phobic patients' perceptions of their dental fear and experiences in dental care. The study sample consisted of 18 participants (12 women), with a mean age of 39.4 years, selected consecutively from patients applying for treatment at a specialized dental fear clinic in Göteborg, Sweden. Dental fear, assessed by the Dental Anxiety Scale, showed score levels well over established levels for severe dental fear. The method for sampling and analysis was inspired by the constant comparative method for Grounded Theory (GT). The thematized in‐depth interviews took place outside the clinic and lasted for 1–1.5 h. All the interviews were conducted by the first author (KHA), audiotaped and transcribed verbatim. Three higher‐order categories were developed and labelled existential threat, vulnerability and unsupportive dentist. Existential threat was identified as the core category, describing the central meaning of the subjects' experiences in dental care. The core category included two dimensions, labelled threat of violation and threat of loss of autonomy and independence. The core category and the descriptive categories are integrated in a model framing the process of dental fear, as described by the informants. In conclusion, the onset of dental fear was commonly related to individual vulnerability and to traumatic dental care experiences, where perceived negative dentist behaviour played a significant role. The patient was caught in a `vicious circle' that was difficult to break, and where fear and anxiety were maintained by negative expectations about treatment and about patient's own ability to cope in dental care situations.
Characterization of new microsatellite loci from Vitis vinifera and their conservation in some Vitis species and hybrids
Authors: Lefort F. , Kyvelos CH. J. , Zervou M. , Edwards K. J. , Roubelakis‐Angelakis K. A.
Published: March 2003

We present here characterization data for seven new microsatellite markers designed from new microsatellite loci isolated from a microsatellite‐enriched DNA library from Vitis vinifera. The observed heterozygosity varied from 0.73 up to 0.93 and the number of alleles per locus ranged from 12 to 26. This high polymorphism makes these new markers interesting for use in genotyping studies and completing the set of microsatellite markers already available for V. vinifera. Additionally these seven new markers appear to be conserved in four other Vitis species and 15 Vitis hybrids used as rootstocks for V. vinifera cultivation.
Isolation and characterization of microsatellite loci in the Galápagos Opuntia weevil Gerstaeckeria galapagoensis (Coleoptera, Curculionidae)
Authors: Dhuyvetter Hilde , Verdyck Peter , Gaublomme Eva , Desender Konjev , Mondor‐Genson Guenaelle , Rasplus Jean‐Yves
Published: March 2003

Five microsatellite loci were isolated from the Galápagos weevil Gerstaeckeria galapagoensis. Polymorphism ranged from two to seven alleles, and observed and expected heterozygosities ranged from 0.286 to 0.917 and 0.254 to 0.683, respectively.