bluuebunny/crossref_metadata_2025 huggingface dataset:
UDP‐N‐acetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellular distribution of cytosolic and nuclear enzyme
Authors: Okuyama Ryo , Marshall Stephen
Published: July 2010

In brain tissue, UDP‐N‐acetylglucosaminyl transferase (OGT) is known to catalyze the addition of a single N‐acetylglucosamine moiety (GlcNAc) onto two proteins linked to the etiology of neurodegenerative disease –β‐amyloid associated protein and tau. Hyperphosphorylation of tau appears to cause neurofibrillary tangles and cell death, and a functional relationship appears to exist between phosphorylation and glycosylation. Since a greater understanding of brain OGT may provide new insights into the pathogenesis of Alzheimer's disease, we examined the characteristics and subcellular distribution of OGT protein and OGT activity and its relationship to O‐linked glycosylation. We found that cytosolic OGT activity is 10 times more abundant in brain tissue compared with muscle, adipose, heart, and liver tissue. Temperature studies demonstrated that cytosolic OGT activity was stable at 24°C but was rapidly inactivated at 37°C (T1/2 = 20 min). Proteases were probably not involved because OGT immunopurified from cytosol retained temperature sensitivity. Subcellular distribution studies showed abundant OGT protein in the nucleus that was enzymatically active. Nuclear OGT activity exhibited a high affinity for UDP‐GlcNAc and a salt sensitivity that was similar to cytosolic OGT; however, nuclear OGT was not inactivated at 37°C, as was the cytosolic enzyme. Two methods were used to measure O‐linked glycoproteins in brain cytosol and nucleosol –[3H]galactose labeling and western blotting using antibodies against O‐linked glycoproteins. Both methods revealed a greater abundance of O‐linked glycoproteins in the nucleus compared to cytosol.
Comparison of circadian expression of tryptophan hydroxylase isoform mRNAs in the rat pineal gland using real‐time PCR
Authors: Sugden David
Published: July 2010

A second gene encoding a functional tryptophan hydroxylase activity has recently been described (TPH2), which is expressed abundantly in brainstem, the primary site of serotonergic neurons in the CNS. As serotonin (5‐HT) has an important role as a precursor of the nocturnal synthesis of the pineal gland hormone, melatonin, it was of interest to determine the relative expression of TPH1 and 2 mRNA in the rat pineal during the light:dark (L:D) cycle using sensitive real‐time RT‐PCR assays which were developed for each TPH isoform. TPH1 mRNA expression was 105‐fold more abundant in rat pineal than TPH2, and showed a significant ∼4‐fold nocturnal increase in expression which may contribute to the previously described nocturnal increase in pineal tryptophan hydroxylase activity. TPH2 expression within the gland showed no significant variation with time of day and was very low (∼300 copies/gland) indicating expression in the small proportion of ‘non‐pinealocyte’ cells in the gland.
Differential profile of CRF receptor distribution in the rat stomach and duodenum assessed by newly developed CRF receptor antibodies
Authors: Chatzaki Ekaterini , Murphy Brian J. , Wang Lixin , Million Mulugeta , Ohning Gordon V. , Crowe Paul D. , Petroski Robert , Taché Yvette , Grigoriadis Dimitri E.
Published: July 2010

Peripheral corticotropin‐releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat upper GI. Polyclonal antisera directed against the C‐terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF1‐ and CRF2‐transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti‐CRF1antiserum (4467a‐CRF1) was identified and used in parallel with another antiserum recognizing both CRF1and CRF2(4392a‐CRF1&2) to immunostain gastric tissue sections. Antiserum 4467a‐CRF1demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus. Conversely, 4392a‐CRF1&2labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre‐absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co‐localization of 4392a‐CRF1&2but not 4467a‐CRF1immunoreactivity with H/K‐ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a‐CRF1&2showed modest immunoreactivity in the duodenal mucosa. Finally, co‐localization of CRF2and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.
4‐Hydroxytamoxifen attenuates methamphetamine‐induced nigrostriatal dopaminergic toxicity in intact and gonadetomized mice
Authors: Kuo Yu‐Min , Chen Hsiang‐Hua , Shieh Chih‐Chang , Chuang Kuo‐Pin , Cherng Chianfang G. , Yu Lung
Published: July 2010

The present study was undertaken to assess the ability of 4‐hydroxytamoxifen (4‐OHT) to alter methamphetamine‐induced nigrostriatal dopaminergic toxicity. Three daily doses of 4‐OHT (6 µg/day) effectively attenuated methamphetamine‐induced nigrostriatal dopamine depletions in both sexes of intact and gonadectomized C57BL/6 J mice. 4‐OHT alone did not alter the dopamine content levels in the striatum. Both male and female mice exhibited similar Cu, Zn‐superoxide dismutase protein levels in the striata whether after gonadectomy or 4‐OHT treatment. Furthermore, basal body temperature and methamphetamine‐induced hyperthermia were not affected by 4‐OHT treatment in either sex of mice. Using a lucigenen‐derived chemiluminescence assay, we found that 4‐OHT by itself can serve as a potent superoxide anion radical scavengerin vitro. The protective effects of 4‐OHT against methamphetamine‐induced nigrostriatal dopamine depletion can be, in part, due to its antioxidative characteristics. The free radical‐scavenging ability of 4‐OHT calls for further investigations for its uses in clinical practice.
Effect of methylphenidate on dopamine/DARPP signalling in adult, but not young, mice
Authors: Fukui Ryuichi , Svenningsson Per , Matsuishi Toyojiro , Higashi Hideho , Nairn Angus C. , Greengard Paul , Nishi Akinori
Published: July 2010

Methylphenidate (MPH), a dopamine uptake inhibitor, is the most commonly prescribed drug for the treatment of attention‐deficit/hyperactivity disorder (ADHD) in children. We examined the effect of MPH on dopamine‐ and cAMP‐regulated phosphoprotein,Mr32 kDa (DARPP‐32) phosphorylation at Thr34 (PKA‐site) and Thr75 (Cdk5‐site) using neostriatal slices from young (14–15‐ and 21–22‐day‐old) and adult (6–8‐week‐old) mice. MPH increased DARPP‐32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice. The effect of MPH was blocked by a dopamine D1 antagonist, SCH23390. In slices from young mice, MPH did not affect DARPP‐32 phosphorylation. As with MPH, cocaine stimulated DARPP‐32 Thr34 phosphorylation in slices from adult, but not from young mice. In contrast, a dopamine D1 agonist, SKF81297, regulated DARPP‐32 phosphorylation comparably in slices from young and adult mice, as did methamphetamine, a dopamine releaser. The results suggest that dopamine synthesis and the dopamine transporter are functional at dopaminergic terminals in young mice. In contrast, the lack of effect of MPH in young mice is likely attributable to immature development of the machinery that regulates vesicular dopamine release.
Enhancement of ischemia‐induced tyrosine phosphorylation of Kv1.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3‐kinase
Authors: Qiu Mei‐Hong , Zhang Rong , Sun Feng‐Yan
Published: July 2010

Our studies observed that, consistent with the literature, ischemic/hypoxic insults increased the expression of voltage‐gated potassium channel (Kv) 1.2 potassium channel as well as elevating the endogenous level of vascular endothelial growth factor (VEGF) in neurons of adult rat brain following middle cerebral artery occlusion and in SH‐SY5Y cells after hypoxia and glucose deprivation. Concomitantly, we also observed that ischemic injury increased the tyrosine phosphorylation of Kv 1.2 inin vivoandin vitro; the introduction of exogenous VEGF could attenuate cell death inin vitromodels. Furthermore, we found that the protective effect of VEGF is mediated through its up‐regulative actions on the tyrosine phosphorylation of Kv 1.2, which in turn has a direct influence on cell viability after ischemic insult. In substantiation of this result, we used anti‐sense methodology to suppress the expression of endogenous VEGF, which significantly inhibited the tyrosine phosphorylation of Kv 1.2 and increased cell death elicited by ischemic/hypoxic injury. Finally, the enhancement of the tyrosine phosphorylation of the channel by VEGF in neuronal cells was significantly attenuated in the presence of wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (PI3‐K), or genestin, an inhibitor of tyrosine kinase, thus suggesting that the phosphorylation of Kv 1.2 induced by VEGF is mechanistically linked to the PI3‐K pathway.
Selective prion protein binding to synaptic components is modulated by oxidative and nitrosative changes induced by copper(II) and peroxynitrite in cholinergic synaptosomes, unveiling a role for calcineurin B and thioredoxin
Authors: Morot‐Gaudry‐Talarmain Y. , Rezaei H. , Guermonprez L. , Treguer E. , Grosclaude J.
Published: July 2010

Choline acetyltransferase (ChAT) and choline transport are decreased after nitrosative stress. ChAT activity is altered in scrapie‐infected neurons, where oxidative stress develops. Cellular prion protein (PrPc) may play a neuroprotective function in participating in the redox control of neuronal environment and regulation of copper metabolism, a role impaired when PrPcis transformed into PrPScin prion pathologies. The complex cross‐talk between PrPcand cholinergic neurons was analyzedin vitrousing peroxynitrite and Cu2+treatments on nerve endings isolated fromTorpedo marmorata, a model of the motoneuron pre‐synaptic element. Specific interactions between solubilized synaptic components and recombinant ovine prion protein (PrPrec) could be demonstrated by Biacore technology. Peroxynitrite abolished this interaction in a concentration‐dependent way and induced significant alterations of neuronal targets. Interaction was restored by prior addition of peroxynitrite trapping agents. Cu2+(in the form of CuSO4) treatment of synaptosomes triggered a milder oxidative effect leading to a bell‐shaped increase of PrPrec binding to synaptosomal components, counteracted by the natural thiol agents, glutathione and thioredoxin. Copper(II)‐induced modifications of thiols in several neuronal proteins. A positive correlation was observed between PrPrec binding and immunoreactive changes for calcineurin B and its partners, suggesting a synergy between calcineurin complex and PrP for copper regulation.
Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3‐kinase activation
Authors: Bijur Gautam N. , Jope Richard S.
Published: July 2010

We describe here a new component of the phosphatidylinositol 3‐kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin‐like growth factor‐1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight‐ to 12‐fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the β‐subunit of ATP synthase and glycogen synthase kinase‐3β. The finding that mitochondrial glycogen synthase kinase‐3β was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase‐3β. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase‐3β in mitochondria.
Repeated cocaine administration changes the function and subcellular distribution of adenosine A1receptor in the rat nucleus accumbens
Authors: Toda Shigenobu , Alguacil Luis F. , Kalivas Peter W.
Published: July 2010

Adenosine A1receptor (A1) protein and mRNA is increased in the nucleus accumbens following repeated cocaine treatment. In spite of this protein up‐regulation, A1agonist‐stimulated [35S]GTPγS binding was attenuated in accumbens homogenates of rats withdrawn for 3 weeks from 1 week of daily cocaine injections. Cellular subfractionation revealed that the discrepancy between total A1protein and G protein coupling resulted from a smaller proportion of receptors in the plasma membrane. The decrease in functional receptor in the plasma membrane was further indicated by diminished formation of heteromeric receptor complex consisting of A1and dopamine D1Areceptors. To explore the functional significance of the altered distribution of A1receptors, at 3 weeks after discontinuing repeated cocaine or saline, animals were injected with cocaine and 45 min later the subcellular distribution of A1receptors quantified. Whereas a cocaine challenge in repeated saline‐treated animals induced a marked increase in membrane localization of the A1receptor, the relative distribution of receptors in repeated cocaine rats was not affected by acute cocaine. These data suggest that the sorting and recycling of A1receptors is dysregulated in the nucleus accumbens as the consequence of repeated cocaine administration.
The mGlu2/3 receptor agonist, LY354740, blocks immobilization‐induced increases in noradrenaline and dopamine release in the rat medial prefrontal cortex
Authors: Swanson Chad J. , Perry Kenneth W. , Schoepp Darryle D.
Published: July 2010

The metabotropic glutamate (mGlu2/3) receptor agonist, LY354740, exhibits anxiolytic‐like properties in a number of rodent models. The present study utilizedin vivomicrodialysis to examine the effects of LY354740 on extracellular monoamine levels in the medial prefrontal cortex (mPFC) of animals subjected to 30 min immobilization stress. Immobilization stress significantly elevated extracellular levels of noradrenaline (NA) and dopamine (DA) in the mPFC, while systemic administration of LY354740 (30 mg/kg, s.c.) significantly attenuated immobilization‐induced increases in both NA and DA. Reverse‐dialysis of LY354740 (30 µm) into the mPFC significantly attenuated immobilization‐induced increases in NA, but not DA without affecting basal levels of either amine. In separate studies in the presence of citalopram (1 µm; reverse dialysis into the mPFC), systemic administration of LY354740 attenuated immobilization‐induced increases in NA and DA, but had no effect on serotonin (5‐HT) levels. Co‐administration of the selective mGlu2/3 receptor antagonist, LY341495, partially or fully reversed the attenuation in NA and DA levels produced by LY354740, respectively. Taken together, these data suggest that LY354740 may produce anti‐stress actions, in part, by blocking stress‐related increases in catecholamines in the mPFC via mGlu2/3 receptor stimulation.
Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of theEAAT1promoter and characterization of its basal and inducible activity in human astrocytes
Authors: Kim Seon‐Young , Choi So‐Young , Chao Wei , Volsky David J.
Published: July 2010

Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1‐kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of theEAAT1gene in human primary astrocytes. The full‐length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor‐binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5′‐deletion analysis of theEAAT1promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between −57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC‐box in position −52/−39 and one E‐box near the TSS and the GC‐box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC‐box and upstream stimulating factor (USF) 1 to the E‐box. Treatment of primary human astrocytes with cellular modulators 8‐bromo cyclic AMP and epidermal growth factor increasedEAAT1promoter activity in transient transfection assays and increased cellularEAAT1mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor‐α reduced bothEAATpromoter activity and cellularEAAT1mRNA expression. These results enable studies of transcriptional regulation ofEAAT1gene at the promoter level.
Different effects of five dopamine receptor subtypes on nuclear factor‐κB activity in NG108‐15 cells and mouse brain
Authors: Takeuchi Yusuke , Fukunaga Kohji
Published: July 2010

We previously showed that dopamine receptors D1R and D2R expressed in NG108‐15 cells activated protein kinase A and extracellular signal‐regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)‐κB activity. To investigate whether other dopamine receptor subtypes regulate NF‐κB, we established NG108‐15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF‐κB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF‐κB‐Luci activity, although forskolin‐induced cyclic AMP responsive element‐Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element‐luciferase reporter not observed following stimulation with quinpirole in D3R‐ or D4R‐expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF‐κB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF‐κB activity. By contrast, risperidone treatment enhanced NF‐κB activity. Taken together, D2R and D1R/D5R had opposite effects on NF‐κB activity in NG108‐15 cells. Risperidone up‐regulated and haloperidol down‐regulated NF‐κB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone.
Expression of a novel neuropeptide, NVGTLARDFQLPIPNamide, in the larval and adult brain of Drosophila melanogaster
Authors: Verleyen Peter , Baggerman Geert , Wiehart Ursula , Schoeters Eric , Van Lommel Alfons , De Loof Arnold , Schoofs Liliane
Published: July 2010

Advances in mass spectrometry and the availability of genomic databases made it possible to determine the peptidome or peptide content of a specific tissue. Peptidomics by nanoflow capillary liquid chromatography tandem mass spectrometry of an extract of 50 larval Drosophila brains, yielded 28 neuropeptides. Eight were entirely novel and encoded by five not yet annotated genes; only two genes had a homologue in the Anopheles gambiae genome. Seven of the eight peptides did not show relevant sequence homology to any known peptide. Therefore, no evidence towards the physiological role of these ‘orphan’ peptides was available. We identified one of the eight peptides, IPNamide, in an extract of the Drosophila adult brain as well. Next, specific antisera were raised to reveal the distribution pattern of IPNamide and other peptides from the same precursor, in larval and adult brains by means of whole‐mount immunocytochemistry and confocal microscopy. IPNamide immunoreactivity is abundantly present in both stages and a striking similarity was found between the distribution patterns of IPNamide and TPAEDFMRFamide, a member of the FMRFamide peptide family. Based on this distribution pattern, IPNamide might be involved in phototransduction, in processing sensory stimuli, as well as in controlling the activity of the oesophagus.
Signalling pathways for insulin‐like growth factor type 1‐mediated expression of uncoupling protein 3
Authors: Gustafsson Helena , Tamm Christoffer , Forsby Anna
Published: July 2010

Uncoupling protein 3 (UCP3) is a mitochondrial protein with antioxidant properties and its regulation by factors promoting cell‐survival may be important for protection of, for instance, neurons in states of oxidative stress. In the present study, we investigated regulatory pathways for UCP3 expression mediated by the neuroprotective hormone insulin‐like growth factor type 1 (IGF‐1) in human neuroblastoma SH‐SY5Y cells. Northern blot analysis and RT‐PCR showed that treatment with 10 nm IGF‐1 increased the UCP3 mRNA levels 2.5‐fold after 5 h. Co‐incubation with the phosphatidylinositol 3 (PI3)‐kinase inhibitor LY294002 prohibited IGF‐1‐mediated induction of both UCP3 mRNA and protein in a concentration‐dependent manner, with a complete blockage at 1 μm, as shown by RT‐PCR and western blot analyses. The mitogen‐activated protein (MAP) kinase kinase 1 (MKK1 or MEK) inhibitor PD98059 also decreased the UCP3 mRNA expression at 10 μm, however, this concentration only partly inhibited the protein expression. We conclude that IGF‐1 enhanced UCP3 expression at transcriptional level, primarily through the PI3‐kinase‐dependent pathway and partly through the MAP kinase pathway.
Tumour necrosis factor alpha inhibits purinergic calcium signalling in blood–brain barrier endothelial cells
Authors: Vandamme Wouter , Braet Katleen , Cabooter Liesbet , Leybaert Luc
Published: July 2010

The breaching of the blood–brain barrier is an essential aspect in the pathogenesis of neuroinflammatory diseases, in which tumour necrosis factor alpha (TNF‐α) as well as endothelial calcium ions play a key role. We investigated whether TNF‐α could influence the communication of calcium signals between brain endothelial cells (GP8 and RBE4). Intercellular calcium waves triggered by mechanical stimulation or photoliberation of InsP3 in single cells were significantly reduced in size after TNF‐α exposure (1000 U/mL, 2 and 24 h). Calcium signals are communicated between cells by means of gap junctional and paracrine purinergic signalling. TNF‐α significantly inhibited gap junctional coupling, stimulated the basal release of ATP, and dose‐dependently blocked the triggered component of ATP release. The cytokine displayed similar effects on the uptake of a fluorescent reporter dye into the cells. Previous work with connexin mimetic peptides demonstrated that the triggered ATP release in these cells is connexin‐related; these peptides did, however, not influence the elevated basal ATP release caused by TNF‐α. We conclude that TNF‐α depresses calcium signal communication in blood–brain barrier endothelial cells, by reducing gap junctional coupling and by inhibiting triggered ATP release. The cytokine thus inhibits connexin‐related communication pathways like gap junctions and connexin hemichannels.
Phosphatidylinositol 3′‐OH kinase and protein kinase A pathways mediate the anti‐apoptotic effect of pituitary adenylyl cyclase‐activating polypeptide in cultured cerebellar granule neurons: modulation by ethanol
Authors: Bhave Sanjiv V. , Hoffman Paula L.
Published: July 2010

Cerebellar granule neurons cultured in the presence of 5 mmKCl undergo spontaneous apoptosis, which is reduced by exposure to pituitary adenylyl cyclase‐activating polypeptide (PACAP). Previous work has suggested roles for the cyclic AMP/PKA and MAP kinase signaling pathways in the anti‐apoptotic effect of PACAP. In the present study, the use of specific inhibitors confirmed the role of the cyclic AMP/PKA pathway, and also demonstrated a role for the phosphatidylinositol 3′‐OH kinase (PI 3‐kinase) neuroprotective pathway in the action of PACAP. Ethanol exposure accelerates the anti‐apoptotic effect of PACAP by a mechanism that involves the PKA and PI‐3 kinase pathways. The results demonstrate that ethanol canincrease neuroprotectioninduced by PACAP. As previous work has shown that ethanol canincrease apoptosisof cerebellar granule neurons by inhibiting the protective effect of agents such as NMDA or IGF‐1, the overall effect of ethanol on cerebellar neuron apoptosis during development may reflect the balance between inhibition and enhancement of the actions of various endogenous neuroprotective agents.
Identification of differentially regulated transcripts in mouse striatum following methamphetamine treatment – an oligonucleotide microarray approach
Authors: Thomas David M. , Francescutti‐Verbeem Dina M. , Liu Xiuli , Kuhn Donald M.
Published: July 2010

Methamphetamine is an addictive drug of abuse that can produce neurotoxic effects in dopamine nerve endings of the striatum. The purpose of this study was to identify new genes that may play a role in the highly complex cascade of events associated with methamphetamine intoxication. Using Affymetrix oligonucleotide arrays, 12 488 genes were simultaneously interrogated and there were 152 whose expression levels were changed following methamphetamine treatment. The genes are grouped into broad functional categories with inflammatory/immune response elements, receptor/signal transduction components and ion channel/transport proteins among the most populated. Many genes within these categories can be linked to ion regulation and apoptosis, both of which have been implicated in methamphetamine toxicity, and numerous factors associated with microglial activation emerged with significant changes in expression. For example, brain‐derived neurotrophic factor (BDNF), chemokine (C‐C) receptor 6 (CCr6) and numerous chemokine transcripts were increased or decreased in expression more than 2.8‐fold. These results point to activated microglia as a potential source of the reactive oxygen/nitrogen species and cytokines that have been previously associated with methamphetamine toxicity and other neurotoxic conditions.
Hypoxia increases calcium flux through cortical neuron glutamate receptors via protein kinase C
Authors: Bickler P. E. , Fahlman C. S. , Ferriero D. M.
Published: July 2010

The effects of 30 s to 10 min hypoxia (PO2−10 mmHg) on glutamate receptor activity were studied in murine cortical neurons. Receptor activity was assessed as a rise in intracellular calcium concentration ([Ca2+]i) following a 10 s application of 1 mmglutamate or 100 µmN‐methy‐d‐aspartate (NMDA) in the presence of 0.1 mmMg2+and 10 µmglycine. Change in [Ca2+]ielicited by glutamate increased 26% (n = 192,p < 0.001) and that to NMDA by 74% (n = 9,p < 0.01) during a 100‐s period of hypoxia. After 10 min hypoxia, responses to glutamate were 62% smaller than those in normoxia, with increased basal intracellular [Ca2+]ipredicting reduced receptor activity. When neurons were exposed to NMDA after 10 min of hypoxia, [Ca2+]iincreases were 12% smaller than after 100 s hypoxia, but still 53% larger than in oxygenated neurons (n = 9,p = 0.01). Neurons expressed relatively similar amounts of NR2A, ‐B, ‐C, and ‐D subunits. The phosphorylation of NMDA NR1 subunits increased during hypoxia. Pre‐treatment of neurons with a protein kinase C (PKC) inhibitor (chelerythrine, 10 µm) prevented increases inN‐methy‐d‐aspartate receptor (NMDAR) activity during hypoxia and reduced the phosphorylation of NR1 subunits. These results suggest that enhancement of glutamate receptor activity during the first minutes of hypoxia is mediated by phosphorylation of NMDARs by PKC and that other mechanisms, possibly involving intracellular calcium, limit glutamate receptor‐mediated calcium influx during longer periods of hypoxia.
Stimulus‐specific plasticity of prefrontal cortex dopamine neurotransmission
Authors: Jackson Mark E. , Moghaddam Bita
Published: July 2010

Future planning and behavioral modification is thought to require experience‐dependent plasticity in neuronal circuits involving the prefrontal cortex, nucleus accumbens and amygdala. Dopamine has been implicated in such plasticity; however, the nature of the adaptive response of dopamine systems to emotionally salient experiences is poorly understood. We determined whether the dopaminergic response to a given stimulus changes after the first exposure to that stimulus and whether this alteration is stimulus specific. Dopamine release was measured in the prefrontal cortex and the nucleus accumbens in response to two aversive but qualitatively distinct stimuli, physical restraint and electrical microstimulation of basolateral amygdala. In the prefrontal cortex, the first exposure to restraint or amygdala stimulation produced similar increases in dopamine release. The second exposure to restraint resulted in an attenuated response (− 36%) whereas the second exposure to amygdala stimulation produced a potentiated response (+ 110%). Cross‐modal potentiation of response occurred with both stimuli. These adaptive changes were specific to the prefrontal cortex and were not observed in the nucleus accumbens. These findings demonstrate that prefrontal cortical dopamine output adapts after a single exposure to stimuli with emotional salience. The direction of this adaptation, however, is not uniform and depends on the nature of the stimulus.
Glycogen metabolism in the rat retina
Authors: Coffe Víctor , Carbajal Raymundo C. , Salceda Rocío
Published: July 2010

It has been reported that glycogen levels in retina vary with retinal vascularization. However, the electrical activity of isolated retina depends on glucose supply, suggesting that it does not contain energetic reserves. We determined glycogen levels and pyruvate and lactate production under various conditions in isolated retina. Ex vivo retinas from light‐ and dark‐adapted rats showed values of 44 ± 0.3 and 19.5 ± 0.4 nmol glucosyl residues/mg protein, respectively. The glycogen content of retinas from light‐adapted animals was reduced by 50% when they were transferred to darkness. Glycogen levels were low in retinas incubated in glucose‐free media and increased in the presence of glucose. The highest glycogen values were found in media containing 20 mm of glucose. A rapid increase in lactate production was observed in the presence of glucose. Surprisingly, glycogen levels were the lowest and lactate production was also very low in the presence of 30 mm glucose. Our results suggest that glycogen can be used as an immediate accessible energy reserve in retina. We speculate on the possibility that gluconeogenesis may play a protective role by removal of lactic acid.
Microglia/macrophage‐specific protein Iba1 binds to fimbrin and enhances its actin‐bundling activity
Authors: Ohsawa Keiko , Imai Yoshinori , Sasaki Yo , Kohsaka Shinichi
Published: July 2010

Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage‐specific calcium‐binding protein. Iba1 has the actin‐bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1‐related intracellular signalling pathway in greater detail, we employed a yeast two‐hybrid screen to isolate an Iba1‐interacting molecule and identified another actin‐bundling protein, L‐fimbrin. In response to stimulation, L‐fimbrin accumulated and co‐localized with Iba1 in membrane ruffles induced by M‐CSF‐stimulation and phagocytic cups formed by IgG‐opsonized beads in microglial cell line MG5. L‐fimbrin was shown to associate with Iba1 in cell lysate of COS‐7 expressing L‐fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L‐fimbrin was demonstrated by immunoprecipitation, glutathione S‐transferase pull‐down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin‐bundling activity of L‐fimbrin. These results indicate that Iba1 forms complexes with L‐fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co‐operates with L‐fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.
Enhanced morphine withdrawal and µ‐opioid receptor G‐protein coupling in A2Aadenosine receptor knockout mice
Authors: Bailey Alexis , Davis Lianne , Lesscher Heidi M. B. , Kelly Mary D. W. , Ledent Catherine , Hourani Susanna M. O. , Kitchen Ian
Published: July 2010

Much evidence supports the hypothesis that A2Aadenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild‐type and A2Areceptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, µ‐opioid and dopamine D2receptor autoradiography, as well as µ‐opioid receptor‐stimulated guanylyl 5′‐[γ‐[35S]thio]‐triphosphate ([35S]GTPγS) autoradiography was carried out in brain sections of withdrawn wild‐type and knockout mice. No significant changes in D2and µ‐opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of µ receptor‐stimulated [35S]GTPγS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2Areceptor plays a role in opioid withdrawal related to functional receptor activation.
Adaptive responses in hypothalamic neuropeptide Y in the face of prolonged high‐fat feeding in the rat
Authors: Hansen Michelle J. , Jovanovska Valentina , Morris Margaret J.
Published: July 2010

While a dysregulation in neuropeptide Y (NPY) signaling has been described in rodent models of obesity, few studies have investigated the time‐course of changes in NPY content and responsiveness during development of diet‐induced obesity. Therefore we investigated the effect of differing lengths (2–17 weeks) of high‐fat diet on hypothalamic NPY peptide content, release and NPY‐induced hyperphagia. Male Sprague–Dawley rats (211 ± 3 g) were fed either a high‐fat diet (30% fat) or laboratory chow (5% fat). Animals were implanted with intracerebroventricular cannulae to investigate feeding responses to NPY (0.5 nmol, 1 nmol) after 4 or 12 weeks of diet. At the earlier stage of obesity, NPY‐induced hyperphagia was not altered; however, animals maintained on the high‐fat diet for the longer duration were hyper‐responsive to NPY, compared to chow‐fed control rats (p < 0.05). Overall, hypothalamic NPY peptide content tended to be decreased from 9 to 17 weeks of diet (p < 0.05). Total hypothalamic NPY content was negatively correlated with plasma leptin concentration (p < 0.05), suggesting the hypothalamic NPY system remains responsive to leptin's inhibitory signal. In addition, hypothalamic NPY overflow was significantly reduced in high‐fat fed animals (p < 0.05). Together these results suggest a reduction in hypothalamic NPY activity in high‐fat fed animals, perhaps in an attempt to restore energy balance.
Down‐regulation of rat brain adenosine A1receptors at the end of pregnancy
Authors: León David , Albasanz José Luis , Fernández Mercedes , Ruíz Maria Angeles , Martín Mairena
Published: July 2010

The status of the adenosine A1receptor/adenylyl cyclase (A1R/AC) transduction pathway in rat brain was analysed at the end of pregnancy using different approaches. Pregnancy at term caused a significant decrease in the Bmaxvalue obtained by saturation binding assays using [3H]DPCPX as radioligand, suggesting a down‐regulation of adenosine A1receptor. Moreover, A1receptor immunodetection in pregnant rat membranes and the level of mRNA coding A1receptor were significantly decreased. This loss of A1receptor was associated with a significant increase in receptor affinity, since the KDvalue from the [3H]DPCPX saturation curve and Kifor N6‐cyclohexyladenosine (CHA) were decreased in pregnant rats. Surprisingly, CHA‐mediated inhibition of adenylyl cyclase was increased, reflecting enhanced receptor responsiveness. On the other hand, immunoblotting of different αGi‐protein isoforms revealed a significant increase in αGi3level in membranes from pregnant rats. Pre‐incubation of membranes with anti‐αGi3antibody blocked the guanosine triphosphate (GTP) or CHA inhibitory effect on adenylyl cyclase in both pregnant and non‐pregnant rats, pointing to αGi3as the main isoform involved in the A1receptor response. These results suggest that, at the end of pregnancy, there is a down‐regulation of adenosine A1 receptors counterbalanced with a strengthened functionality, probably due to an increase in both αGi3protein and receptor affinity.
Cell type‐dependent recruitment of trichostatin A‐sensitive repression of the human 5‐HT1A receptor gene
Authors: Lemonde Sylvie , Rogaeva Anastasia , Albert Paul R.
Published: July 2010

Regulation of serotonin (5‐HT)1A receptor expression in brain is implicated in mood disorders such as depression and anxiety. Transcriptional activity of the human 5‐HT1A receptor gene was strongly repressed by a negative regulatory region containing a consensus repressor element‐1 (RE‐1) and two copies of the dual repressor element (DRE) identified in the rat 5‐HT1A receptor gene. REST/NRSF, a silencer of neuronal genes, bound the 5‐HT1A RE‐1 and repressed the 5‐HT1A promoter. Inactivation of RE‐1 completely abolished REST‐mediated repression, but resulted in only partial (15–50%) de‐repression of basal 5‐HT1A promoter activity. The human 5‐HT1A DRE sequences bound specifically to the novel repressor Freud‐1 (5′repressor element under dual repression binding protein‐1) and conferred repressor activity at 5‐HT1A or SV40 promoters. In 5‐HT1A‐negative cells [L6, human embryonic kidney (HEK) 293], the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) abolished repression mediated by both RE‐1/REST and DRE/Freud‐1, and induced almost complete de‐repression of the 5‐HT1A gene. By contrast, in 5‐HT1A‐expressing neuronal cells (RN46A, SN‐48) TSA blocked RE‐1/REST repression, but did not affect DRE/Freud‐1‐mediated repression. Thus in contrast to REST, Freud‐1 mediates HDAC‐independent repression of the 5‐HT1A receptor promoter in neuronal 5‐HT1A‐positive cells, suggesting that HDAC recruitment might influence neuron‐specific gene expression by further silencing expression in non‐neuronal tissue.
Regulation of β‐amyloid precursor protein expression by brain‐derived neurotrophic factor involves activation of both the Ras and phosphatidylinositide 3‐kinase signalling pathways
Authors: Ruiz‐León Yolanda , Pascual Angel
Published: July 2010

Brain‐derived neurotrophic factor (BDNF) stimulates β‐amyloid precursor protein (APP) promoter activity by a Ras‐dependent mechanism in TrkB‐expressing SH‐SY5Y cells. To determine the signalling pathways involved in the BDNF‐induced response, we have analysed the ability of TrkB mutated forms to mediate promoter stimulation. Brain‐derived neurotrophic factor causes a significant induction of promoter activity and mutation K540R in the active site of TrkB completely abolishes the neurotrophin‐induced response. A substitution of the Y484 residue by phenylalanine, which blocks binding of Shc, reduces the activation of APP promoter by BDNF by approximately 50% whereas mutation Y785P, which blocks binding of phospholipase C gamma, does not affect the response. In addition, the phosphatidylinositide 3‐kinase (PI3K)‐specific inhibitors wortmannin and LY294002 reduced BDNF‐induced activation. In agreement with a participation of both Ras/MAPK‐ and PI3K/Akt‐mediated mechanisms, transient expression of constitutive active forms of Ras, PI3K and other components of both signalling pathways led to a significant increase of APP promoter activity. Furthermore, the stimulation of the APP promoter by BDNF was completely precluded by expression of dominant‐negative forms of Ras and PI3K effectors. Taken together, our results suggest that simultaneous activation of at least two signalling pathways, Ras/MAPK and PI3K/Akt, is necessary to mediate a full activation of the APP promoter by BDNF.
Protein associated with Myc (PAM) is involved in spinal nociceptive processing
Authors: Ehnert Corina , Tegeder Irmgard , Pierre Sandra , Birod Kerstin , Nguyen Hong‐Van , Schmidtko Achim , Geisslinger Gerd , Scholich Klaus
Published: July 2010

PAM (protein associated with Myc) is a potent inhibitor of adenylyl cyclases (ACs) which is primarily expressed in neurones. Here we describe that PAM is highly expressed in dorsal horn neurones and motoneuron of the spinal cord, as well as in neurones of dorsal root ganglia in adult rats. PAM mRNA expression is differentially regulated during development in both spinal cord and dorsal root ganglia of rats, being strongest during the major respective synaptogenic periods. In adult rats, PAM expression was up‐regulated in the spinal cord after peripheral nociceptive stimulation using zymosan and formalin injection, suggesting a role for PAM in spinal nociceptive processing. Since PAM inhibited Gαs‐stimulated AC activity in dorsal root ganglia as well as spinal cord lysates, we hypothesized that PAM may reduce spinal nociceptive processing by inhibition of cAMP‐dependent signalling. Accordingly, intrathecal treatment with antisense but not sense oligonucleotides against PAM increased basal and Gαs‐stimulated AC activity in the spinal cord and enhanced formalin‐induced nociceptive behaviour in adult rats. Taken together our findings demonstrate that PAM is involved in spinal nociceptive processing.
Overexpression of torsinA in PC12 cells protects against toxicity
Authors: Shashidharan P. , Paris Nicolae , Sandu Daniela , Karthikeyan Laina , McNaught Kevin St P. , Walker Ruth H. , Olanow C. Warren
Published: July 2010

Childhood‐onset dystonia is an autosomal dominant movement disorder associated with a three base pair (GAG) deletion mutation in the DYT1 gene. This gene encodes a novel ATP‐binding protein called torsinA, which in the central nervous system is expressed exclusively in neurons. Neither the function of torsinA nor its role in the pathophysiology of DYT1 dystonia is known. In order to better understand the cellular functions of torsinA, we established PC12 cell lines overexpressing wild‐type or mutant torsinA and subjected them to various conditions deleterious to cell survival. Treatment of control PC12 cells with an inhibitor of proteasomal activity, an oxidizing agent, or trophic withdrawal, resulted in cell death, whereas PC12 cells that overexpressed torsinA were significantly protected against each of these treatments. Overexpression of mutant torsinA failed to protect cells against trophic withdrawal. These results suggest that torsinA may play a protective role in neurons against a variety of cellular insults.
Up‐regulation of glutamate concentration in the putamen and in the prefrontal cortex of asymptomatic SIVmac251‐infected macaques without major brain involvement
Authors: Bossuet Christophe , Vaufrey Françoise , Condé Françoise , Chrétien Fabrice , Pichon Jacques , Hantraye Philippe , Le Grand Roger , Dormont Dominique , Gras Gabriel
Published: July 2010

We quantified putamen and prefrontal cortex metabolites in macaques with simian immunodeficiency virus infection and searched for virological and histological correlates. Fourteen asymptomatic macaques infected since 8–78 months (median: 38) were compared with eight uninfected ones. Absolute concentrations of acetate, alanine, aspartate, choline, creatine, GABA, glutamate, glutamine, lactate, myo‐inositol,N‐acetylaspartate, taurine and valine were determined byex vivoproton magnetic resonance spectroscopy. Glutamate concentration in the CSF was determined by HPLC. Gliosis was assessed by glial fibrillary acidic protein and CD68 immunohistochemistry. Glutamate concentration was slightly increased in the prefrontal cortex (19%,p = 0.0152,t‐test) and putamen (13%,p = 0.0354,t‐test) of the infected macaques, and was unaffected in the CSF. Myo‐inositol concentration was increased in the prefrontal cortex only (27%,p = 0.0136). The concentrations of glutamate and myo‐inositol in the prefrontal cortex were higher in the animals with marked or intense microgliosis (p = 0.0114). The other studied metabolites, includingN‐acetylaspartate, were not altered. Glutamate concentration may thus increase in the cerebral parenchyma in asymptomatic animals, but is not accompanied by a detectable decrease inN‐acetylaspartate concentration (neuronal dysfunction). Thus, there are probably compensatory mechanisms that may limit glutamate increase and/or counterbalance its effects.
Early anti‐inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats
Authors: Bao Feng , Chen Yuhua , Dekaban Gregory A. , Weaver Lynne C.
Published: July 2010

We investigated mechanisms by which a monoclonal antibody (mAb) against the CD11d subunit of the leukocyte integrin CD11d/CD18 improves neurological recovery after spinal cord injury (SCI) in the rat. The effects of an anti‐CD11d mAb treatment were assessed on ED‐1 expression (estimating macrophage infiltration), myeloperoxidase activity (MPO, approximating neutrophil infiltration), lipid peroxidation, inducible nitric oxide synthase (iNOS) and nitrotyrosine (indicating protein nitration) expression in the spinal cord lesion after severe clip‐compression injury. Protein expression was evaluated by western blotting and immunocytochemistry. Lipid peroxidation was assessed by thiobarbituric acid reactive substances (TBARS) production. After anti‐CD11d mAb treatment, decreased ED‐1 expression at 6–72 h after SCI indicated reduced macrophage infiltration. MPO activity (units/g tissue) was reduced significantly from 114 ± 11 to 75 ± 8 (− 34%) at 6 h and from 38 ± 2 to 22 ± 4 (− 42%) at 72 h. After SCI, anti‐CD11d mAb treatment significantly reduced TBARS from 501 ± 61 to 296 ± 17 nm (− 41%) at 6 h and to approximately uninjured values (87 nm) at 72 h. The mAb treatment also attenuated the expression of iNOS and formation of nitrotyrosine at 6–72 h after SCI. These data indicate that anti‐CD11d mAb treatment blocks intraspinal neutrophil and macrophage infiltration, reducing the intraspinal concentrations of reactive oxygen and nitrogen species. These effects likely underlie improved tissue preservation and neurological function resulting from the mAb treatment.