bluuebunny/crossref_metadata_2025 huggingface dataset:
Amphetamine and D1 Dopamine Receptor Agonists Produce Biphasic Effects on Glutamate Efflux in Rat Ventral Tegmental Area: Modification by Repeated Amphetamine Administration
Authors: Wolf Marina E. , Xue Chang‐Jiang
Published: July 2010

Abstract: Amphetamine or selective D1 and D2 dopamine receptor agonists and antagonists were administered to the ventral tegmental area (VTA) through a microdialysis probe to determine their effects on glutamate and aspartate efflux in rats pretreated for 5 days with vehicle or 5 mg/kg (+)‐amphetamine sulfate. In vehicle rats, glutamate efflux declined during 2 h of perfusion with the D1 receptor agonist SKF‐82958 (10 and 100 µM). After SKF‐82958 perfusion ended, glutamate efflux rebounded to basal levels and continued to increase gradually over the next 2 h. A similar biphasic pattern was observed with intra‐VTA amphetamine (10 and 100 µM) and with another D1 agonist (100 µM SKF‐38393). The biphasic effects of SKF‐82958 were prevented by coperfusion with a D1 antagonist (SCH‐23390; 30 µM). Glutamate efflux was unaffected by a D2 agonist (100 µM quinpirole) and by D1 or D2 antagonists administered alone (SCH 23390 and eticlopride; 30 µM). In amphetamine‐pretreated rats tested 2 days after the last injection, both the decrease during SKF‐82958 perfusion and the delayed increase in glutamate efflux were attenuated. In rats tested 12–14 days after the last amphetamine injection, only the decrease during SKF‐82958 perfusion was attenuated. None of these drug treatments produced consistent effects on aspartate efflux. We showed previously that systemic amphetamine (5 mg/kg, i.p.) has no immediate effect on VTA glutamate efflux but produces a delayed increase in glutamate efflux that reaches statistical significance 2–3 h after injection. Because behavioral sensitization can be elicited either by repeated systemic or repeated intra‐VTA administration, neurochemical effects common to both routes (such as the delayed increase in glutamate efflux) are most likely to contribute to its induction.
Effects of α‐Phenyl‐tert‐Butylnitrone and Selegiline on Hydroxyl Free Radicals in Rat Striatum Produced by Local Application of Glutamate
Authors: Ferger B. , Van Amsterdam C. , Seyfried C. , Kuschinsky K.
Published: July 2010

Abstract: The hydroxyl radical is a very reactive oxygen species that damages biomolecules in the brain and in other tissues. The possible pharmacological intervention to prevent hydroxyl radical formation was studied in vivo using the microdialysis technique in brains of nonanesthetized rats. Hydroxyl radicals form stable adducts [mainly 2,3‐dihydroxybenzoic acid (2,3‐DHBA) and 2,5‐DHBA)] via an aromatic hydroxylation reaction with salicylic acid. 2,3‐DHBA was separated and quantified by HPLC and electrochemical detection. Microdialysis probes were implanted into the striatum 1 day before measurement of levels of hydroxyl radicals. The next day, the probes were first perfused for 120 min with a modified Ringer's solution containing 5 mM salicylic acid, to obtain stable baselines. Afterward, the perfusion solution was switched to another solution that in addition contained 50 mM glutamate, to stimulate radical formation. Twenty minutes later, α‐phenyl‐tert‐butylnitrone (PBN; 100 mg/kg), selegiline (10 mg/kg), or saline was administered intraperitoneally. The glutamate perfusion produced marked two‐ to 2.5‐fold increases in 2,3‐DHBA content. Treatment with PBN significantly antagonized the rise of 2,3‐DHBA level, indicating that PBN is a direct radical scavenger not only in vitro but also in vivo. Acute treatment with selegiline failed to reduce significantly the glutamate‐induced radical formation. The acute experiments presented here do not support the suggestion that the neuroprotective effects of selegiline described in the literature are due to a potential hydroxyl radical scavenging property of the drug.
Congo Red Inhibits Proteoglycan and Serum Amyloid P Binding to Amyloid β Fibrils
Authors: Gupta‐Bansal Rekha , Brunden Kurt R.
Published: July 2010

Abstract: Various data suggest that Alzheimer's disease results from the accumulation of amyloid β (Aβ) peptide fibrils and the consequent formation of senile plaques in the cognitive regions of the brain. One approach to lowering senile plaque burden in Alzheimer's disease brain is to identify compounds that will increase the degradation of existing amyloid fibrils. Previous studies have shown that proteoglycans and serum amyloid P (SAP), molecules that localize to senile plaques, bind to Aβ fibrils and protect the amyloid peptide from proteolytic breakdown. Therefore, molecules that prevent the binding of SAP and/or proteoglycans to fibrillar Aβ might increase plaque degradation and prove useful in the treatment of Alzheimer's disease. The nature of SAP and proteoglycan binding to Aβ is defined further in the present study. SAP binds to both fibrillar and nonfibrillar forms of Aβ. However, only the former is rendered resistant to proteolysis after SAP association. It is interesting that both SAP and proteoglycan binding to Aβ fibrils can be inhibited by glycosaminoglycans and Congo red. Unexpectedly, Congo red protects fibrillar Aβ from breakdown, suggesting that this compound and other structurally related molecules are unlikely to be suitable for use in the treatment of Alzheimer's disease.
3‐Hydroxykynurenine, an Endogenous Oxidative Stress Generator, Causes Neuronal Cell Death with Apoptotic Features and Region Selectivity
Authors: Okuda Shoki , Nishiyama Nobuyoshi , Saito Hiroshi , Katsuki Hiroshi
Published: July 2010

Abstract: 3‐Hydroxykynurenine (3‐HK) is a potential endogenous neurotoxin whose increased levels have been described in several neurodegenerative disorders. Here, we characterized in vitro neurotoxicity of 3‐HK. Of the tested kynurenine pathway metabolites, only 3‐HK, and to a lesser extent 3‐hydroxyanthranilic acid, were toxic to primary cultured striatal neurons. 3‐HK toxicity was inhibited by various antioxidants, indicating that the generation of reactive oxygen species is essential to the toxicity. 3‐HK‐induced neuronal cell death showed several features of apoptosis, as determined by the blockade by macromolecule synthesis inhibitors, and by the observation of cell body shrinkage with nuclear chromatin condensation and fragmentation. In addition, 3‐HK toxicity was dependent on its cellular uptake via transporters for large neutral amino acids, because uptake inhibition blocked the toxicity. Cortical and striatal neurons were much more vulnerable to 3‐HK toxicity than cerebellar neurons, which may be attributable to the differences in transporter activities of these neurons. These results indicate that 3‐HK, depending on transporter‐mediated cellular uptake and on intracellular generation of oxidative stress, induces neuronal cell death with brain region selectivity and with apoptotic features, which may be relevant to pathology of neurodegenerative disorders.
Cyclin D2 Interacts with cdk‐5 and Modulates Cellular cdk‐5/p35 Activity
Authors: Guidato Sonia , McLoughlin Declan M. , Grierson Andrew J. , Miller Christopher C. J.
Published: July 2010

Abstract: Cyclin‐dependent kinase‐5 (cdk‐5) is a serine/threonine kinase that displays neurone‐specific activity. Experimental manipulation of cdk‐5 expression in neurones has shown that cdk‐5 is essential for proper development of the nervous system and, in particular, for outgrowth of neurites. Such observations suggest that cdk‐5 activity must be tightly controlled during development of the nervous system. To identify possible regulators of cdk‐5, we used the yeast two‐hybrid system to search for proteins that interact with cdk‐5. In two independent yeast transformation events, cyclin D2 interacted with cdk‐5. Immunoprecipitation experiments confirmed that cyclin D2 and cdk‐5 interact in mammalian cells. Cyclin D2 did not activate cdk‐5 as assayed using three different substrates, which was in contrast to a known cdk‐5 activator, p35. However, cyclin D2 expression led to a decrease in cdk‐5/p35 activity in transfected cells. As cyclin D2 and cdk‐5 are known to share overlapping patterns of expression during development of the CNS, the results presented here suggest a role for cyclin D2 in modulating cdk‐5 activity in postmitotic developing neurones.
Contributions of N‐Linked Glycosylation to the Expression of a Functional α7‐Nicotinic Receptor in Xenopus Oocytes
Authors: Chen DaNong , Dang Hong , Patrick James W.
Published: July 2010

Abstract: The α7 subunit of the neuronal nicotinic acetylcholine receptor, when expressed in Xenopus oocytes, forms homooligomeric ligand‐gated ion channels that are blocked by a snake toxin, α‐bungarotoxin. The amino‐terminal extracellular domain of the α7 sequence has three consensus sites for asparagine‐linked glycosylation (N46DS, N90MS, and N133AS). In this study, we show that α7 expressed either in vivo or in vitro is a glycoprotein of 57 kDa. In addition, we demonstrate by site‐directed mutagenesis that all three consensus sites are used for glycosylation. To elucidate the role(s) of asparagine‐linked glycosylation in the formation and function of the α7 receptor, wild‐type and glycosylation‐deficient α7 subunits were expressed in COS cells and oocytes. We examined biochemical and physiological properties of expressed receptors and found that α7 glycosylation mutations do not affect homooligomerization and surface protein expression of the α7 receptor but do affect surface expression of α‐bungarotoxin binding sites and the function of the receptor. Our data indicate that asparagine‐linked glycosylation is required for the expression of a functional α7 receptor in oocytes.
AT1 Receptor‐Mediated Nuclear Translocation of Raf‐1 in Brain Neurons
Authors: Lu Di , Yang Hong , Raizada Mohan K.
Published: July 2010

Abstract: Angiotensin II (Ang II) interacts with the neuronal AT1 receptor subtype and initiates a cascade of signaling events involving activation of Ras‐Raf‐1‐MAP kinase. Raf‐1‐dependent activation of mitogen‐activated protein kinase (MAPK) is the key in the chronic norepinephrine neuromodulatory actions of Ang II and is associated with the translocation of MAPK into the nucleus. In view of these observations, this study was designed to determine if Ang II causes cellular redistribution of Raf‐1 in neuronal cells. Most of Raf‐1 was localized in the cytoplasmic compartment in neurons. Ang II treatment resulted in a time‐dependent increase in the translocation of immunoreactive Raf‐1 from the cytoplasm into the nucleus. A fourfold increase was observed in 15 min. The nuclear sequestration of Raf‐1 was blocked by losartan, an AT1 receptor‐specific antagonist, and not by PD123319, an AT2 receptor‐specific antagonist. Confocal microscopic analysis of immunofluorescence data confirmed the nuclear translocation and further showed that Raf‐1 was exclusively localized into the nucleolus. These observations demonstrate, for the first time, that Ang II stimulates Raf‐1 targeting into the neuronal nucleus, and they suggest that this translocation may play a direct role in the transcriptional regulation of Ang II actions.
Trimethyltin Stimulates Protein Kinase C Translocation Through Receptor‐Mediated Phospholipase C Activation in PC12 Cells
Authors: Kane Michael D. , Yang Ching‐Wen , Gunasekar Palur G. , Isom Gary E.
Published: July 2010

Abstract: Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT‐induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT‐stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5‐trisphosphate (IP3), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)‐α‐methyl‐4‐carboxyphenylglycine; (+)‐MCPG]. Furthermore, the rise in IP3 level was blocked by pretreating cells with a PLC inhibitor (U‐73122) or by a combination of atropine and (+)‐MCPG. This pretreatment also significantly decreased TMT‐stimulated PKC translocation, indicating that TMT‐mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)‐MCPG did not provide protection against TMT‐induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein‐coupled receptors, ultimately leading to PKC translocation.
Peripherin Is Tyrosine‐Phosphorylated at Its Carboxyl‐Terminal Tyrosine
Authors: Angelastro James M. , Ho Chung‐Liang , Frappier Thierry , Liem Ronald K. H. , Greene Lloyd A.
Published: July 2010

Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf‐9 cells and SW‐13 cl. 2 vim− cells. The identity of peripherin as a tyrosine‐phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two‐dimensional isoelectric focusing/sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW‐13 cl. 2 vim− cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy‐terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild‐type peripherin and peripherin Y474F form a filamentous network in SW‐13 cl. 2 vim− cells. This indicates that tyrosine phosphorylation of the peripherin C‐terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.
Proenkephalin Gene Regulation in the Paraventricular Nucleus by GABA: Interactions with Opioid Systems in a Transgenic Model
Authors: Borsook David , Falkowski Olga , Smirnova Olga
Published: July 2010

Abstract: GABA, which is present at high levels within the paraventricular nucleus (PVN) of the hypothalamus, has been implicated in neuroendocrine regulation. Here we use a transgenic mouse model expressing a human proenkephalin‐β‐galactosidase fusion gene, in which both up‐regulation and down‐regulation of gene expression can be measured easily, to study the effects of GABA on basal and stress‐induced gene expression within the PVN. This model has been shown previously to be appropriately physiologically regulated by stress within the PVN. Acute systemic administration of GABA, of aminooxyacetic acid, and of the selective GABAB receptor agonist, baclofen, induces transgene expression in the PVN. Chronic administration of aminooxyacetic acid inhibits both basal and stress‐induced transgene expression. Acute or chronic administration of the selective GABAA agonist, muscimol, inhibits both basal and stress‐induced expression of the transgene. Muscimol also inhibits transgene expression in hypothalamic slice cultures in which extrahypothalamic afferents are removed. It is surprising that, following chronic aminooxyacetic acid administration, the opiate antagonist naltrexone induces transgene expression in a manner similar to that observed with naloxone‐precipitated opioid withdrawal and that expression is accompanied by a behavioral syndrome that partially mimicks opioid withdrawal. Taken together with our previous work, and using gene expression as our read‐out, we conclude that transgene‐expressing PVN neurons receive tonic inhibitory inputs mediated via GABAA receptors. Moreover, these inhibitory inputs can themselves be inhibited via GABAB and μ‐opioid receptors.
Hydrogen Peroxide Inhibits the Vacuolar H+‐ATPase in Brain Synaptic Vesicles at Micromolar Concentrations
Authors: Wang Yalin , Floor Erik
Published: July 2010

Abstract: Hydrogen peroxide (H2O2) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H2O2 on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H+‐ATPase (V‐ATPase) in the vesicle membrane. We report here that the V‐ATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H2O2. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H2O2‐induced inhibition of the V‐ATPase, and ATP or GTP partially protect the ATPase from inhibition by H2O2. These and other results suggest that the mechanism of inhibition of the V‐ATPase by H2O2 involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V‐ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down‐regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.
Nicotine‐Stimulated Release of [3H]Norepinephrine from Fetal Rat Locus Coeruleus Cells in Culture
Authors: Gallardo Kathy A. , Leslie Frances M.
Published: July 2010

Abstract: Acute nicotine administration stimulated [3H]norepinephrine ([3H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC50 of 0.9 µM, and was abolished by removal of calcium from, or addition of tetrodotoxin (500 nM) to, the assay buffer. Other nicotinic receptor agonists stimulated [3H]NE release, with the rank order of potency being (±)‐epibatidine > (−)‐nicotine > 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP). Whereas (−)‐nicotine and (±)‐epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (−)‐cytisine had no agonist activity. Nicotine‐stimulated release of [3H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro‐β‐erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an α4β2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA‐ and nicotine‐stimulated [3H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK‐801 blockade of nicotine‐induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [3H]NE from LC cells and does not act indirectly via activation of glutamate release.
Na+‐Dependent and Phlorizin‐Inhibitable Transport of Glucose and Cycasin in Brain Endothelial Cells
Authors: Matsuoka Toshiyuki , Nishizaki Tomoyuki , Kisby Glen E.
Published: July 2010

Abstract: Although cycasin (methylazoxymethanol β‐d‐glucoside) is proposed to be a significant etiological factor for the prototypical neurodegenerative disorder Western Pacific amyotrophic lateral sclerosis and parkinsonism‐dementia complex, the mechanism underlying transport of cycasin across the blood‐brain barrier (BBB) is unknown. We examined cycasin transport in cultured bovine brain endothelial cells, a major element of the BBB. Cycasin was taken up into endothelial cells in a dose‐dependent manner with maximal uptake observed at a concentration of 10 µM. Cycasin uptake was significantly inhibited by α‐methyl‐d‐glucoside, a specific analogue for the Na+‐dependent glucose transporter (SGLT), by the SGLT inhibitor phlorizin, by replacement of extracellular NaCl with LiCl, and by dinitrophenol (DNP), an inhibitor of energy metabolism. In addition, cycasin produced inward currents in a whole‐cell voltage clamp configuration. Peak currents were observed at 10 µM with a trend toward reduction at higher concentrations, and currents were clearly blocked by α‐methyl‐d‐glucoside, phlorizin, and DNP. In addition, cycasin never evoked currents in Na+‐free extracellular solution. These results suggest that cycasin is selectively transported across brain endothelial cells, possibly across the BBB by a Na+/energy‐dependent glucose transporter.
Phospholipid‐Metabolizing Enzymes in Alzheimer's Disease: Increased Lysophospholipid Acyltransferase Activity and Decreased Phospholipase A2 Activity
Authors: Ross Brian M. , Moszczynska Anna , Erlich Jeffrey , Kish Stephen J.
Published: July 2010

Abstract: Damage to brain membrane phospholipids may play an important role in the pathogenesis of Alzheimer's disease (AD); however, the critical metabolic processes responsible for the generation and repair of membrane phospholipids affected by the disease are unknown. We measured the activity of key phospholipid catabolic and anabolic enzymes in morphologically affected and spared areas of autopsied brain of patients with AD and in matched control subjects. The activity of the major catabolic enzyme phospholipase A2 (PLA2), measured in both the presence and absence of Ca2+, was significantly decreased (−35 to −53%) in parietal and temporal cortices of patients with AD. In contrast, the activities of lysophospholipid acyltransferase, which recycles lysophospholipids into intact phospholipids, and glycerophosphocholine phosphodiesterase, which returns phospholipid catabolites to be used in phospholipid resynthesis, were increased by ∼50–70% in the same brain areas. Brain activities of enzymes involved in de novo phospholipid synthesis (ethanolamine kinase, choline kinase, choline phosphotransferase, phosphoethanolamine cytidylyltransferase, and phosphocholine cytidylyltransferase) were either normal or only slightly altered. The activities of PLA2 and acyltransferase were normal in the degenerating cerebellum of patients with spinocerebellar atrophy type 1, whereas the activity of glycerophosphocholine phosphodiesterase was reduced, suggesting that the alterations in AD brain were not nonspecific consequences of neurodegeneration. Our data suggest that compensatory phospholipid metabolic changes are present in AD brain that reduce the rate of phospholipid loss via both decreased catabolism (PLA2) and increased phospholipid resynthesis (acyltransferase and glycerophosphocholine phosphodiesterase).
Lasting Increase in Serotonin 5‐HT1A but Not 5‐HT4 Receptor Subtypes in the Kindled Rat Dentate Gyrus: Dissociation from Local Presynaptic Effects
Authors: Cagnotto A. , Crespi D. , Mancini L. , Manzoni C. , Presti M. L. , Gariboldi M. , Vezzani A. , Mennini T.
Published: July 2010

Abstract: We examined the effect of kindling on serotonergic neurotransmission in the hippocampus by measuring serotonin (5‐HT) release and uptake in hippocampal synaptosomes and 5‐HT1A and 5‐HT4 receptor subtypes during and at different times after electrical kindling of the dentate gyrus. Using quantitative receptor autoradiography, we found that binding of 8‐[3H]hydroxy‐2‐(di‐n‐propylamino)tetralin ([3H]8‐OH‐DPAT) to 5‐HT1A receptors was selectively increased by 20% on average (p < 0.05) in the dentate gyrus of the stimulated and contralateral hippocampus 2 days after stage 2 (stereotypes and occasional retraction of a forelimb) and by 100% on average (p < 0.05) 1 week after stage 5 (tonic‐clonic seizures) compared with sham‐stimulated rats. A 20% increase (p < 0.05) was observed 1 month after the last generalized seizure. No changes were found after a single afterdischarge. 5‐HT4 receptors, which colocalize with 5‐HT1A receptors on hippocampal neurons, were not modified in kindled tissue. [3H]5‐HT uptake and its release as well as the 5‐HT1B autoreceptor function did not differ from shams in hippocampal synaptosomes at stages 2 and 5. Systemic administration of 100 and 1,000 µg kg−1 8‐OH‐DPAT or 1,000 µg kg−1 WAY‐100,635, 30 min before each electrical stimulation, did not significantly alter kindling progression or the occurrence of stage 5 seizures in fully kindled rats. The changes in 5‐HT1A receptor density in the dentate gyrus are part of the plastic modifications occurring during kindling and may contribute to modulating tissue hyperexcitability.
Use‐Dependent Suppression of the Nicotinic Acetylcholine Receptor Response by the Proadrenomedullin N‐Terminal 20‐Amino Acid Peptide in Rat Locus Coeruleus Neurons
Authors: Nabekura Junichi , Murata Osamu , Ishibashi Hitoshi , Akaike Norio
Published: July 2010

Abstract: The effects of the proadrenomedullin N‐terminal 20‐amino acid peptide (PAMP) on the nicotinic acetylcholine (ACh) receptor (nAChR)‐mediated inward current were investigated in neurons acutely dissociated from the rat locus coeruleus using whole‐cell recording under voltage clamp. Nicotine and cytidine mimicked the ACh response, whereas the maximal response to dimethyl‐phenylpiperazinium was lower in amplitude compared with that to ACh. Nicotine‐induced current (Inic) was suppressed more effectively by mecamylamine than by hexamethonium. In addition, neither atropine nor α‐bungarotoxin affected the Inic. PAMP reversibly and noncompetitively suppressed the peak amplitude of 10−4MInic. PAMP concentrations for the threshold, half‐maximal inhibition, and maximal inhibition of 10−4M Inic were 10−8, 2.6 × 10−7, and 10−5M, respectively. The peak amplitudes of 10−4M Inic elicited at 2‐min intervals showed a gradual decline in the presence of 10−7M PAMP. This decline in the Inic was independent of the period of PAMP pretreatment. The suppression of Inic by PAMP did not show any voltage dependency at a holding potential (VH) of <0 mV, although the inhibitory effect was masked by the marked inward rectification of Inic at a VH of 0 mV. These results suggest that PAMP could thus be a unique endogenous peptide that antagonizes the nAChR in the CNS.
Light‐Dependent Expression of Pinopsin Gene in Chicken Pineal Gland
Authors: Takanaka Yoko , Okano Toshiyuki , Iigo Masayuki , Fukada Yoshitaka
Published: July 2010

Abstract: The phase of a circadian clock in the chicken pineal gland is reset by an environmental light signal, which is captured by the pineal photoreceptive molecule(s). Here we show that the mRNA level of pinopsin, a predominant photoreceptive molecule in the pineal gland, undergoes a diurnal fluctuation in chickens maintained on a light/dark cycle. The mRNA levels in the light were approximately six times higher than those in the dark. This fluctuation was not observed in constant darkness, where the mRNA levels remained low. Subsequent light exposure of chickens increased the amount of pinopsin mRNA regardless of the circadian time. Clearly, the expression of pinopsin gene is controlled by a light signal, independent of the circadian clock. In vitro experiments using cultured pineal glands isolated from the visual system also revealed the light‐dependent increase in pinopsin mRNA level, indicating that the pineal photoreceptive molecule(s) is responsible for the induction. These results demonstrate the presence of a feedback loop through which the light signal captured by pinopsin stimulates the transcription of its own gene in the chicken pineal gland. In contrast, pinopsin protein remained at an almost constant level in chickens maintained under the same light/dark cycles. The protein level, however, decreased to ∼50% of the light/dark level under constant darkness and subsequently increased upon exposure to light after the dark period. It is suggested that, under the light/dark cycles, the pinopsin protein level is kept constant by the light‐dependent synthesis, which would compensate for a possible degradation of pinopsin in the daytime.
Stimulation of p42 and p44 Mitogen‐Activated Protein Kinases by Reactive Oxygen Species and Nitric Oxide in Hippocampus
Authors: Kanterewicz Beatriz I. , Knapp Lauren T. , Klann Eric
Published: July 2010

Abstract: Reactive oxygen species (ROS) have been suggested to act as cellular messengers that mediate signal transduction cascades in various cell types. However, little is known about their role in this capacity in the nervous system. We have begun to investigate the role of ROS, and that of nitric oxide (NO), in mediating mitogen‐activated protein kinase (MAPK) signaling in rat hippocampal slices. Our studies have revealed that direct exposure of hippocampal slices to hydrogen peroxide, xanthine/xanthine oxidase (a superoxide‐generating system), sodium nitroprusside (an NO donor compound), S‐nitroso‐N‐acetylpenicillamine (an NO donor compound), or 3‐morpholinosydnonimine (a compound that produces NO and superoxide) results in an enhancement in tyrosine phosphorylation of several proteins, including proteins with apparent molecular masses of 42 and 44 kDa. We investigated the possibility that these proteins correspond to the active forms of p42 MAPK and p44 MAPK. Hippocampal slices exposed to various ROS and NO donors resulted in increases in levels of the active forms of both p42 MAPK and p44 MAPK. The ROS‐ and NO‐enhanced tyrosine phosphorylation and activation of p42 MAPK and p44 MAPK were inhibited by pretreatment with the antioxidant N‐acetyl‐l‐cysteine. Our observations indicate that ROS and NO can mediate protein tyrosine phosphorylation and MAPK signaling in the hippocampus via a redox‐sensitive mechanism and suggest a potential cellular mechanism for their effects in the nervous system.
Dominant Negative ATF1 Blocks Cyclic AMP‐Induced Neurite Outgrowth in PC12D Cells
Authors: Shimomura Atsushi , Okamoto Yoshichika , Hirata Yoko , Kobayashi Makoto , Kawakami Kiyoshi , Kiuchi Kazutoshi , Wakabayashi Takashi , Hagiwara Masatoshi
Published: July 2010

Abstract: Extension of the neuronal process is a crucial step for establishment of the neuronal network. As CREB preferentially forms heterodimers with ATF1 in PC12D cells, we examined the roles of the CREB/ATF1 heterodimer on cyclic AMP (cAMP)‐induced neurite extension, using originally constructed ATF1RL, which has a point mutation at the DNA binding domain of ATF1. Transient expression of ATF1RL suppressed the protein kinase A/CREB‐induced expression of the CRE reporter gene as expected. Treatment with forskolin elicited a relatively poor mRNA induction for immediate early genes in PC12D‐ATF1RL cells, a PC12D cell line stably expressing ATF1RL, in comparison with the parental PC12D cells. Furthermore, the PC12D‐ATF1RL cells were proved to be defective at cAMP‐induced neurite outgrowth. In contrast, both the gene expression and the differentiation after nerve growth factor treatment noted in PC12D‐ATF1RL cells were at the same levels as those in the parental cells. These data provide us the first evidence that links CREB/ATF1 to the cAMP‐induced differentiation of PC12 cells.
Local Activation of Metabotropic Glutamate Receptors Inhibits the Handling‐Induced Increased Release of Dopamine in the Nucleus Accumbens but Not that of Dopamine or Noradrenaline in the Prefrontal Cortex: Comparison with Inhibition of Ionotropic Receptors
Authors: Feenstra Matthijs G. P. , Botterblom Margriet H. A. , Van Uum Johanna F. M.
Published: July 2010

Abstract: On‐line in vivo microdialysis was used to determine the effects of a 16‐min handling period on release of dopamine (DA) in the nucleus accumbens and of DA and noradrenaline (NA) in the medial prefrontal cortex of awake, freely moving rats. DA and NA were determined in one HPLC run. Handling resulted in an immediate and strong increase of both catecholamines in the prefrontal cortex. Maximal values for DA were 295%, and for NA 225%, of controls. DA in the nucleus accumbens was also increased (to 135% of controls) but only after a short delay. Local inhibition of ionotropic glutamate receptors by continuous reversed dialysis of the drugs 6‐cyano‐7‐nitroquinoxaline, d‐2‐amino‐5‐phosphonopentanoic acid, or dizocilpine did not significantly affect handling‐induced increases in cortical DA and NA release. Neither did the agonist of metabotropic glutamate receptors, trans‐(1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACPD), or the GABA‐B agonist baclofen. Reversed dialysis of dizocilpine in the nucleus accumbens was equally ineffective, but ACPD inhibited the increase in DA release in this area. Stimulation of metabotropic glutamate receptors in the nucleus accumbens was previously reported to inhibit activation of DA release in that area after stimulation of glutamatergic or dopaminergic afferents. It is concluded that metabotropic receptors in the nucleus accumbens are important for the control of activation of DA release in the accumbens by physiological stimuli but that a similar mechanism is lacking in the prefrontal cortex.
Chronic Ethanol Treatment Decreases [3H]Epibatidine and [3H]Nicotine Binding and Differentially Regulates mRNA Levels of Nicotinic Acetylcholine Receptor Subunits Expressed in M10 and SH‐SY5Y Neuroblastoma Cells
Authors: Gorbounova Olga , Svensson Anne‐Lie , Jönsson Patrik , Mousavi Malahat , Miao Hui , Hellström‐Lindahl Eva , Nordberg Agneta
Published: July 2010

Abstract: For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing α4β2 nicotinic receptor subtype and an SH‐SY5Y neuroblastoma cell line expressing α3, α5, α7, β2, and β4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose‐related decrease (−30–80%) in number of [3H]nicotine binding sites was observed in ethanol‐treated (25–240 mM) compared with untreated M10 cells. Similarly, 4‐day treatment with ethanol in concentrations relevant to chronic alcoholism (100 mM) decreased the number of nicotinic receptor binding sites in the SH‐SY5Y cells when measured using [3H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up‐regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 mM ethanol significantly decreased the mRNA level for the α3 nAChR subunit (−39%), while the mRNA levels for the α7 (+30%) and α4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the β2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.
Multidrug Resistance‐Related Transport Proteins in Isolated Human Brain Microvessels and in Cells Cultured from These Isolates
Authors: Seetharaman Seeta , Barrand Margery A. , Maskell Lynn , Scheper Rik J.
Published: July 2010

Abstract: The multidrug transporter, P‐glycoprotein (Pgp), at the blood‐brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothelium or on adjacent astrocyte foot processes. In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance‐associated protein (MRP), is compared with that for the endothelial marker, platelet‐endothelial cell adhesion molecule‐1 (PECAM‐1) and for the astrocyte‐derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels. Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular [3H]vincristine accumulation. The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM‐1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT‐PCR encoding only Pgp and PECAM‐1, but not GFAP or MRP. Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adherent astrocyte foot processes. In cells grown from the microvessels, although PECAM‐1 remains, Pgp expression decreases and MRP appears. Evidence suggests these multidrug transporters are functionally active in the cultured cells.
Differential Regulation of GABAA Receptor Gene Expression by Ethanol in the Rat Hippocampus Versus Cerebral Cortex
Authors: Matthews Douglas B. , Devaud Leslie L. , Fritschy J. M. , Sieghart W. , Morrow A. Leslie
Published: July 2010

Abstract: Previous research has shown that chronic ethanol consumption dramatically alters GABAA receptor α1 and α4 subunit gene expression in the cerebral cortex and GABAA receptor α1 and α6 subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABAA receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABAA receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABAA receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABAA receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABAA receptor α4 subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABAA receptor α1, α2, α3, α2/3, or γ2 was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABAA receptor α4 subunits and significantly decreased the relative expression of cerebral cortical GABAA receptor α1 subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABAA receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.
Ascorbate‐Induced Oxidative Inactivation of Zn2+‐Glycerophosphocholine Cholinephosphodiesterase
Authors: Sok Dai‐Eun
Published: July 2010

Abstract: Zn2+‐glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38°C. The inclusion of copper ions or Fe2+ accelerated the ascorbate‐induced inactivation, with Cu2+ or Cu+ being much more effective than Fe2+, suggestive of ascorbate‐mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H2O2 inactivated the enzyme in a concentration‐dependent manner. Also, the enzyme was inactivated partially by a superoxide anion‐generating system but not an HOCl generator. In support of involvement of H2O2 in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous •OH. Although p‐nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu2+‐induced inactivation of the phosphodiesterase was compared with the ascorbate‐mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu2+ action. Here, it is proposed that Zn2+‐glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.
α2‐Macroglobulin Attenuates β‐Amyloid Peptide 1–40 Fibril Formation and Associated Neurotoxicity of Cultured Fetal Rat Cortical Neurons
Authors: Du Yansheng , Bales Kelly R. , Dodel Richard C. , Liu Xiaodong , Glinn Michele A. , Horn Jeffrey W. , Little Sheila P. , Paul Steven M.
Published: July 2010

Abstract: β‐Amyloid peptides (Aβ) are deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of Aβ in cultured neurons is dependent on its aggregation state, but the factors contributing to aggregation and fibril formation are poorly understood. In the present study, we investigated whether α2‐macroglobulin (α2M), a protein present in neuritic plaques and elevated in Alzheimer's disease brain, is a potential regulatory factor for Aβ fibril formation. Previous studies in our laboratory have shown that α2M is an Aβ binding protein. We now report that, in contrast to another plaque‐associated protein, α1‐antichymotrypsin, α2M coincubated with Aβ significantly reduces aggregation and fibril formation in vitro. Additionally, cultured fetal rat cortical neurons are less vulnerable to the toxic actions of aged Aβ following pretreatment with α2M. We postulate that α2M is able to maintain Aβ in a soluble state, preventing fibril formation and associated neurotoxicity.
Autoradiography of Serotonin 5‐HT1A Receptor‐Activated G Proteins in Guinea Pig Brain Sections by Agonist‐Stimulated [35S]GTPγS Binding
Authors: Dupuis Delphine S. , Palmier Christiane , Colpaert Francis C. , Pauwels Petrus J.
Published: July 2010

Abstract: G protein activation mediated by serotonin 5‐HT1A and 5‐HT1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist‐stimulated [35S]GTPγS binding to brain sections. [35S]GTPγS binding was stimulated by the mixed 5‐HT1A/5‐HT1B/D agonist L694247 in brain structures enriched in 5‐HT1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [35S]GTPγS binding in brain regions with high densities of 5‐HT1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [35S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5‐HT1B inverse agonist SB224289 (10 µM) did not affect the L694247‐mediated [35S]GTPγS binding response, and the mixed 5‐HT1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [35S]GTPγS binding response and the antagonist profile suggest the L694247‐mediated response in guinea pig brain to be mediated by 5‐HT1A receptors. In addition to L694247, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, and flesinoxan also stimulated [35S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [35S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [35S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5‐HT1A but not 5‐HT1B/D receptors can be measured in guinea pig brain sections.
Nociceptin/Orphanin FQ Activates Mitogen‐Activated Protein Kinase in Chinese Hamster Ovary Cells Expressing Opioid Receptor‐Like Receptor
Authors: Lou Li‐Guang , Zhang Zhe , Ma Lan , Pei Gang
Published: July 2010

Abstract: The effect of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the newly identified opioid receptor‐like (ORL1) receptor, on mitogen‐activated protein kinase (MAPK) was investigated in Chinese hamster ovary cells stably expressing ORL1 receptor. N/OFQ rapidly stimulated phosphorylation and activity of MAPK (p42 and p44 isoforms) in a concentration‐dependent manner. The p42 isoform was preferentially activated by N/OFQ. Maximal activation (5.4 ± 1.2‐fold of basal for p42 isoform) was achieved after a 1‐min exposure of cells to 100 nM N/OFQ. The activation was blocked completely by pretreatment with pertussis toxin, but was not reversed by naloxone. U‐73122, a phospholipase C‐specific inhibitor, significantly inhibited phospholipase C activity, as well as MAPK activation stimulated by N/OFQ. Furthermore, N/OFQ‐stimulated MAPK activation was suppressed by a protein kinase C‐specific inhibitor, chelerythrine. The results demonstrate that N/OFQ can effectively stimulate MAPK by the activation of ORL1 receptor and pertussis toxin‐sensitive G proteins, and that phospholipase C, as well as protein kinase C, is critically involved in these processes.
A Novel Orphan G Protein‐Coupled Receptor Primarily Expressed in the Brain Is Localized on Human Chromosomal Band 2q21
Authors: Bläsius Rainer , Weber Ruthild G. , Lichter Peter , Ogilvie Adaling
Published: July 2010

Abstract: A human hippocampus cDNA library was screened with a probe obtained from degenerate RT‐PCR aimed at P2Y‐homologous sequences. A positive clone, designated hip4, was identified containing an open reading frame of 1,020 bp that had been previously detected in a published genomic clone called R12. Subsequent screening of a human fetal brain cDNA library yielded a splice variant with a 1,104‐bp open reading frame, which was named fb1. Both variants display the seven‐transmembrane topology that is typical for G protein‐coupled receptors. Probing a human multitissue northern blot revealed two brain‐specific transcripts of 2.3 and 6.3 kb, respectively. Northern blot analyses with specific fragments confirmed that the two transcripts are generated by alternative polyadenylation yielding two different 3′ untranslated regions. A genomic clone from the corresponding gene was isolated and mapped to human chromosomal band 2q21 by fluorescence in situ hybridization.
Characterization of Two Distinct Monoclonal Antibodies Specific for Glial Cell Line‐Derived Neurotrophic Factor
Authors: Xu Ren Y. , Pong Kevin , Yu Yanbin , Chang David , Liu Shuying , Lile Jack D. , Treanor James , Beck Klaus D. , Louis Jean‐Claude
Published: July 2010

Abstract: Here we report the generation and characterization of two distinct monoclonal antibodies, G‐90 and B‐1531, specific to glial cell line‐derived neurotrophic factor (GDNF). ELISA results confirmed that G‐90 and B‐1531 both recognize GDNF. Western blots showed that G‐90 recognized only the GDNF dimer, whereas B‐1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G‐90 and B‐1531 recognize different epitopes: PCE confirmed that B‐1531 binds to NH2‐terminal peptides between amino acids 18 and 37, whereas G‐90 does not; BIAcore data showed that B‐1531 binds to the NH2 terminus of GDNF, whereas G‐90 does not. G‐90, in a concentration‐dependent manner, completely neutralized the GDNF‐induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B‐1531 had no neutralizing effects. GDNF‐induced Ret autophosphorylation in NGR‐38 cells was completely neutralized by G‐90, whereas B‐1531 had a moderate effect. These data show that G‐90 and B‐1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to downregulate GDNF‐induced biological activity.