bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipid Distribution and Fatty Acid Composition in Different Brain Regions During Chick Embryo Development
Authors: Rizzo A. M. , Galli C. F. , Montorfano G. , Berra B.
Published: July 2010

Abstract: The phospholipid profile of different chick embryo brain regions was studied from 11 to 21 days of development, revealing interesting changes in content and distribution. Total phospholipid phosphorus (P), in micrograms of P per microgram of DNA, increases significantly during development of cerebral hemispheres (CHs), optic lobes (OLs), and brainstem (BS). Compared with CH and OL, the BS shows at all stages a significantly higher concentration of phospholipid P, which in contrast decreases in the cerebellum (CB) during development. Moreover, the data show interesting differences between the right and the left portion of the brain. The distribution of phospholipid P and the fatty acid composition of phospholipids were asymmetric between left and right OL and CH, as were the concentrations of DNA and cholesterol, demonstrating lateralized neurochemical development in these structures, i.e., left OL, right OL, left CH, and right CH. The data are discussed also in relation to the potential importance of neurochemical lateralization for determining lateralized embryonic and postnatal behavior of this species.
Differential Regulation of Molecular Subtypes of Muscarinic Receptors in Alzheimer's Disease
Authors: Flynn Donna D. , Ferrari‐DiLeo Gaby , Mash Deborah C. , Levey Allan I.
Published: July 2010

Abstract: Molecular subtypes of muscarinic receptors (m1–m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease. However, the status of these receptors in human brain and Alzheimer's disease is incompletely understood. The m1–m5 receptors in brains from control subjects and Alzheimer's disease patients were examined using a panel of specific antisera and radioligand binding. Quantitative immunoprecipitation demonstrated a predominance of the m1, m2, and m4 receptor subtypes in cortical and subcortical regions in control subjects. In Alzheimer's disease, normal levels of m1 receptors measured by radioligand binding contrasted with decreased m1 receptor immunoreactivity, suggesting that the m1 receptor is altered in Alzheimer's disease. The m2 immunoreactivity was decreased, consistent with the loss of m2 binding sites and the location of this receptor subtype on presynaptic cholinergic terminals. The m4 receptor was up‐regulated significantly and may offer a target for new memory‐enhancing drugs. Differential alterations of molecular subtypes of muscarinic receptors may contribute to the cholinergic component of Alzheimer's disease dementia.
Identification of Nuclear Proteins that Are Developmentally Regulated in Embryonic Rat Brain
Authors: Thormodsson Finnbogi R. , Redmond Lori , Hockfield Susan
Published: July 2010

Abstract: To identify nuclear proteins that might play a role in the acquisition of neuronal phenotype, two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) was used to analyze nuclear proteins expressed over the course of embryonic rat brain development. Metabolically labeled rat brain nuclear proteins from embryonic day 14 (E14) were compared with proteins from embryonic day 20 (E20). Over this period, the rat brain develops from a collection of relatively homogeneous precursor cells into a complex structure containing many different classes of neurons. Computer‐assisted analysis of 2D‐PAGE fluorograms identified 11 proteins that show increases in their rate of synthesis between E14 and E20. Twenty proteins that consistently appear at E20 are not detectable on fluorograms of E14 nuclear proteins, even after long exposures, and thus may be considered to appear de novo. Fifty‐eight proteins show consistent down‐regulation between E14 and E20, and of these, 19 are not detectable on fluorograms of E20 nuclear proteins. The electrophoretic properties of many of these proteins suggest that they are previously unreported, developmentally regulated nuclear proteins. Some of the developmentally regulated, brain‐enriched nuclear proteins identified here may play a role in regulating the expression of neural genes important for cellular differentiation in the mammalian CNS.
Interleukin‐2 Transcripts in Human and Rodent Brains: Possible Expression by Astrocytes
Authors: Eizenberg Orly , Faber‐Elman Anat , Lotan Mirit , Schwartz Michal
Published: July 2010

Abstract: Cytokines have been suggested to be involved in the cross talk between the immune and the nervous systems, under normal and pathological conditions. For example, the cytokine interleukin‐2 was suggested to be involved in response to CNS trauma and spontaneous regeneration. Here, we examined whether mammalian CNS has an intrinsic potential to produce interleukin‐2 and, if so, what its cellular origin is. mRNA sequences encoding for interleukin‐2 were detected in brains of humans and rodents. Northern blot analysis revealed the presence of several interleukin‐2 transcripts of different sizes in the brain, all recognized by lymphocyte‐derived interleukin‐2 cDNA probes. One of the transcripts, a high molecular weight form of ∼5 kb, appeared to be unique to the brain. Reverse transcription and amplification by PCR of human fetal brain mRNA revealed one cDNA product that, upon sequence analysis, showed a high degree of homology with the human lymphocyte‐derived interleukin‐2 coding sequence. To identify the possible cellular source of the interleukin‐2 transcripts within the mammalian brain, we similarly analyzed mRNA of rat brain cells in culture. Northern blot analysis revealed that astrocytes contain transcripts that hybridize with interleukin‐2 cDNA probe. These findings point to the astrocytes as a possible source of brain interleukin‐2.
Coupling of the Cloned μ‐Opioid Receptor with the ω‐Conotoxin‐Sensitive Ca2+ Current in NG108‐15 Cells
Authors: Morikawa Hitoshi , Fukuda Kazuhiko , Kato Shigehisa , Mori Kenjiro , Higashida Haruhiro
Published: July 2010

Abstract: Voltage‐dependent Ca2+ currents were measured in NG108‐15 neuroblastoma × glioma hybrid cells transformed to express the rat μ‐opioid receptor by the whole‐cell configuration of the patch‐clamp technique with Ba2+ as charge carrier. A μ‐opioid receptor‐selective agonist, [d‐Ala2, N‐Me‐Phe4,Gly5‐ol]enkephalin caused significant inhibition of voltage‐dependent Ca2+ currents in μ‐receptor‐transformed NG108‐15 cells but not in nontransfected or vector‐transformed control cells. On the other hand, a δ‐opioid receptor‐selective agonist, [d‐penicillamine2,d‐penicillamine5]enkephalin, induced inhibition of voltage‐dependent Ca2+ currents in both control and μ‐receptor‐transformed cells, which is mediated by the δ‐opioid receptor expressed endogenously in NG108‐15 cells. The inhibition of voltage‐dependent Ca2+ currents induced by [d‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin and [d‐penicillamine2,d‐penicillamine5]enkephalin was reduced by pretreatment of the cells with pertussis toxin or ω‐conotoxin GVIA. These results indicate that the μ‐opioid receptor expressed from cDNA functionally couples with ω‐conotoxin‐sensitive N‐type Ca2+ channels through the action of pertussis toxin‐sensitive G proteins in NG108‐15 cells.
Specific Activity of Brain Palmitoyl‐CoA Pool Provides Rates of Incorporation of Palmitate in Brain Phospholipids in Awake Rats
Authors: Grange Eric , Deutsch Joseph , Smith Quentin R. , Chang Michael , Rapoport Stanley I. , Purdon A. David
Published: July 2010

Abstract: In vivo rates of palmitate incorporation into brain phospholipids were measured in awake rats following programmed intravenous infusion of unesterified [9,10‐3H]palmitate to maintain constant plasma specific activity. Animals were killed after 2–10 min of infusion by microwave irradiation and analyzed for tracer distribution in brain phospholipid and phospholipid precursor, i.e., brain unesterified palmitate and palmitoyl‐CoA, pools. [9,10‐3H]Palmitate incorporation into brain phospholipids was linear with time and rapid, with >50% of brain tracer in choline‐containing glycerophospholipids at 2 min of infusion. However, tracer specific activity in brain phospholipid precursor pools was low and averaged only 1.6–1.8% of plasma unesterified palmitate specific activity. Correction for brain palmitoyl‐CoA specific activity increased the calculated rate of palmitate incorporation into brain phospholipids (0.52 nmol/s/g) by ∼60‐fold. The results suggest that palmitate incorporation and turnover in brain phospholipids are far more rapid than generally assumed and that this rapid turnover dilutes tracer specific activity in brain palmitoyl‐CoA pool owing to release and recycling of unlabeled fatty acid from phospholipid breakdown.
Basic Fibroblast Growth Factor Selectively Increases AMPA‐Receptor Subunit GluR1 Protein Level and Differentially Modulates Ca2+ Responses to AMPA and NMDA in Hippocampal Neurons
Authors: Cheng Bin , Furukawa Katsutoshi , O'Keefe Joan A. , Goodman Yadong , Kihiko Muthoni , Fabian Thomas , Mattson Mark P.
Published: July 2010

Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate‐receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration‐dependent increase in levels of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)‐receptor subunit GluR1 protein as determined by western blot, dot‐blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA‐receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium‐imaging studies revealed that elevation of [Ca2+]i, resulting from selective AMPA‐receptor activation, was enhanced in bFGF‐pretreated neurons. On the other hand, [Ca2+]i responses to NMDA‐receptor activation were suppressed in bFGF‐treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate‐receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity‐dependent neuronal plasticity and vulnerability to excitotoxicity.
Functional Reconstitution of a Highly Purified μ‐Opioid Receptor Protein with Purified G Proteins in Liposomes
Authors: Fan Li‐Qun , Gioannini Theresa L. , Wolinsky Toni , Hiller Jacob M. , Simon Eric J.
Published: July 2010

Abstract: A μ‐opioid receptor protein (μ‐ORP) purified to homogeneity from bovine striatal membranes has been functionally reconstituted in liposomes with highly purified heterotrimeric guanine nucleotide regulatory proteins (G proteins). A mixture of bovine brain G proteins, predominantly GoA, was used for most of the experiments, but some experiments were performed with individual pure G proteins, GoA, GoB, Gi1, and Gi2. Low Km GTPase was stimulated up to 150% by μ‐opioid receptor agonists when both μ‐ORP and a G protein (either the brain G protein mixture or a single heterotrimeric G protein) were present in the liposomes. Stimulation by a selective μ‐agonist was concentration dependent and was reversed by the antagonist (−)‐naloxone, but not by its inactive enantiomer, (+)‐naloxone. The μ selectivity of μ‐ORP was demonstrated by the inability of δ and κ agonists to stimulate GTPase in this system. High‐affinity μ‐agonist binding was also restored by reconstitution with the brain G protein mixture and with each of the four pure Gi and Go proteins studied. The binding of μ agonists is sensitive to inhibition by GTPγS and by sodium.
Modulation of Fetal Rat Brain and Liver Phospholipid Content by Intraamniotic Ethyl Docosahexaenoate Administration
Authors: Green Pnina , Yavin Ephraim
Published: July 2010

Abstract: In an attempt to elucidate the role of docosahexaenoic acid (DHA; 22:6n‐3) in the developing brain, a method was devised whereby rapid enrichment of fetal brain and liver lipid with DHA was achieved. Fetal rats at 17 days of gestation were injected intraamniotically with ethyl docosahexaenoate (EtDHA). Control fetuses were administered ethyl oleate (EtOle). Brain lipid DHA content increased by almost 21% (p = 0.02) 3 days after EtDHA administration as compared with EtOle‐injected fetuses, whereas liver lipid DHA content increased by almost 60% (p = 0.0002). At this time brain phosphatidylinositol content doubled, whereas phosphatidylserine (PS) content increased by >50% (p = 0.03). Increases in liver PS (+25.8%; p = 0.015) and sphingomyelin (+43.6%; p = 0.01) content were observed. A redistribution of total brain phospholipid (PL) DHA was observed following Et‐DHA administration, resulting in a 56.4% increase in PS‐DHA abundance (p < 0.05) and an 8.8% decrease in phosphatidylethanolamine‐DHA abundance (p = 0.05). These results suggest modulation of fetal brain and liver PL and provide a method for enrichment of DHA content in discrete PLs during intrauterine life.
N‐System Amino Acid Transport at the Blood‐CSF Barrier
Authors: Keep Richard F. , Xiang Jianming
Published: July 2010

Abstract: Despite l‐glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L‐, A‐, ASC‐, and N‐system amino acid transporters in l‐[14C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na+‐dependent mechanism. The Na+‐independent uptake was inhibited by 2‐amino‐2‐norbornane carboxylic acid, indicating that it is probably via an L‐system transporter. Na+‐dependent uptake was inhibited neither by the A‐system substrate α‐(methylamino)isobutyric acid nor by the ASC‐system substrate cysteine. It was inhibited by histidine, asparagine, and l‐glutamate γ‐hydroxamate, three N‐system substrates. Replacement of Na+ with Li+ had little effect on uptake, another feature of N‐system amino acid transport. These data therefore indicate that N‐system amino acid transport is present at the choroid plexus. The Vmax and Kmax for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 mM, respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.
γ‐Glutamylglutamine and Taurine Concentrations Are Decreased in the Cerebrospinal Fluid of Drug‐Naive Patients with Schizophrenic Disorders
Authors: Do K. Q. , Lauer C. J. , Schreiber W. , Zollinger M. , Gutteck‐Amsler U. , Cuénod M. , Holsboer F.
Published: July 2010

Abstract: HPLC and gas chromatography‐mass spectrometry analyses of 18 amino acids, N‐acetylaspartate, N‐acetylaspartyglutamate, and 5‐hydroxyindoleacetic acid, derived from serotonin, and homovanillic acid, derived from dopamine, were performed in CSF collected from a group of patients with schizophrenia who either had been drug free for at least 1 year (n = 5) or were drug naive for psychotropic drugs (n = 21) and in 15 control subjects. Significant differences were found only for taurine (15% lower in the patients) and isoleucine (7% higher). A number of unidentified substances were detected, one of which proved to be markedly reduced (16%) among the schizophrenic patients. Liquid chromatography‐mass spectrometry with continuous flow‐fast atom bombardment interface allowed us to identify this substance as γ‐glutamyglutamine. The decreased level of γ‐glutamylglutamine may reflect a deficiency in the γ‐glutamyltransferase system, a system probably involved in glutamate uptake, or a deficiency in glutamine, an important precursor of releasable glutamate. Although glutamate was nonsignificantly reduced in the patients, it was one of the five substances (including γ‐glutamylglutamine) that were necessary for the best discrimination between the schizophrenic patients and the controls. These findings support the notion that the glutamatergic system is affected in schizophrenic disorders. In addition, they underscore the need to apply rigid bioanalytical techniques and use drug‐naive patients to gain in‐depth information on the pathophysiology of brain disorders such as schizophrenia.
Immunolabeling of Central Serotonin 5‐HT1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti‐Peptide Antiserum
Authors: Langlois X. , Gérard C. , Darmon M. , Chauveau J. , Hamon M. , El Mestikawy S.
Published: July 2010

Abstract: A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5‐HT1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti‐peptide antibodies were detected by enzyme‐linked immunosorbent assay and characterized by immunoprecipitation of the 5‐HT1B/1Dβ receptor in CHAPS‐solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin‐O‐carboxymethylglycyl[125I]iodotyrosinamide ([125I]GTI; a selective 5‐HT1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5‐HT1B receptors. The remaining 40% of [125I]GTI binding sites were shown to be 5‐HT1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5‐HT1B/1Dβ, but not the 5‐HT1D/1Dα, receptor in transiently transfected LLC‐PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5‐HT1Dβ receptor‐like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [125I]GTI in the brain of these species further confirmed that the anti‐peptide antibodies selectively recognized only the 5‐HT1Dβ component of [125I]GTI specific receptor binding sites.
Effect of MPTP and l‐Deprenyl on Antioxidant Enzymes and Lipid Peroxidation Levels in Mouse Brain
Authors: Thiffault C. , Aumont N. , Quirion R. , Poirier J.
Published: July 2010

Abstract: Excessive free radical formation or antioxidant enzyme deficiency can result in oxidative stress, a mechanism proposed in the toxicity of MPTP and in the etiology of Parkinson's disease (PD). However, it is unclear if altered antioxidant enzyme activity is sufficient to increase lipid peroxidation in PD. We therefore investigated if MPTP can alter the activity of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH‐PX) and the level of lipid peroxidation. l‐Deprenyl, prior to MPTP administration, is used to inhibit MPP+ formation and its subsequent effect on antioxidant enzymes. MPTP induced a threefold increase in SOD activity in the striatum of C57BL/6 mice. No parallel increase in GSH‐PX or CAT activities was observed, while striatal lipid peroxidation decreased. At the level of the substantia nigra (SN), even though increases in CAT activity and reduction in SOD and GSH‐PX activities were detected, lipid peroxidation was not altered. Interestingly, l‐deprenyl induced similar changes in antioxidant enzymes and lipid peroxidation levels, as did MPTP. Taken together, these results suggest that an alteration in SOD activity, without compensatory increases in CAT or GSH‐PX activities, is not sufficient to induce lipid peroxidation.
Phosphorylation of F1F0 ATPase δ‐Subunit Is Regulated by Platelet‐Derived Growth Factor in Mouse Cortical Neurons In Vitro
Authors: Zhang Frank X. , Pan Weiying , Hutchins James B.
Published: July 2010

Abstract: The δ subunit of F1F0 ATPase (ATP synthase complex) is part of the stalk connecting the F1 and F0 moieties. Studies in Escherichia coli suggest that the analogous bacterial subunit, called ε, is essential for the ATPase assembly energy coupling. Platelet‐derived growth factor (PDGF) is an important growth factor for various cell types, including neurons of the CNS. Using two‐dimensional gel electrophoresis, microsequencing, western blot analysis, and immunoprecipitation techniques, we have found that PDGF induces phosphorylation of the δ subunit or a closely related peptide in cultured mouse cortical neurons.
Cloning, Characterization, and Chromosomal Localization of a Human 5‐HT6 Serotonin Receptor
Authors: Kohen Ruth , Metcalf Mark A. , Khan Naseem , Druck Teresa , Huebner Kay , Lachowicz Jean E. , Meltzer Herbert Y. , Sibley David R. , Roth Bryan L. , Hamblin Mark W.
Published: July 2010

Abstract: We describe the cloning and characterization of a human 5‐HT6 serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5‐HT6 cDNA encodes a 440‐amino‐acid polypeptide whose sequence diverges significantly from that published for the rat 5‐HT6 receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5‐HT6 amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5‐HT6 receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5‐HT1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5‐HT6 receptor also reveals an RsaI restriction fragment length polymorphism within the coding region.
Overexpression of Ca2+/Calmodulin‐Dependent Protein Kinase II Inhibits Neurite Outgrowth of PC12 Cells
Authors: Tashima Kazuhiro , Yamamoto Hideyuki , Setoyama Chiaki , Ono Tsunehiko , Miyamoto Eishichi
Published: July 2010

Abstract: To investigate the physiological role of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca2+/calmodulin‐independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP‐dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.
Induction of Adrenal Tyrosine Hydroxylase mRNA by Single Immobilization Stress Occurs Even After Splanchnic Transection and in the Presence of Cholinergic Antagonists
Authors: Kvetňanský Richard , Nankova Bistra , Hiremagalur Bhargava , Viskupic Emil , Vietor Ilja , Rusnak Milan , McMahon Anne , Kopin Irwin J. , Sabban Esther L.
Published: July 2010

Abstract: Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO‐induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold‐induced rise in TH mRNA levels. In this study, the IMO‐induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress‐elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO‐induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.
Homocysteate‐Evoked Release of Acetylcholine from the Rabbit Retina
Authors: Linn David M. , Massey Stephen C.
Published: July 2010

Abstract: The cholinergic amacrine cells of the rabbit retina can be labeled with [3H]choline and the activity of the cholinergic population monitored by following the release of [3H]acetylcholine. It has been proposed that l‐homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [3H]acetylcholine. In magnesium‐free medium, d‐homocysteate was slightly more potent than the l‐isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to l‐homocysteate. 2‐Amino‐7‐phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked l‐homocysteate evoked responses. 6,7‐Dinitroquinoxaline‐2,3‐dione, a potent kainate antagonist, preferentially blocked d‐homocysteate‐evoked responses. Therefore, in the rabbit retina, l‐homocysteate is an NMDA‐preferring agonist, whereas d‐homocysteate is a kainate‐preferring agonist. In addition, we found that l‐homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA‐receptor activation. However, the low potency of l‐homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of l‐homocysteate at the bipolar‐to‐cholinergic amacrine cell synapse.
Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons
Authors: Fontana Giovanni , De Bernardi Roberto , Ferro Federico , Gemignani Anita , Raiteri Maurizio
Published: July 2010

Abstract: l‐Glutamate, NMDA, dl‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin‐like immunoreactivity (SRIF‐LI) from primary cultures of rat hippocampal neurons. In Mg2+‐containing medium, the maximal effects (reached at ∼100 µM) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µM (AMPA), 39 µM (glutamate), 41 µM (KA), and 70 µM (NMDA). The metabotropic receptor agonist trans‐1‐aminocyclopentane‐1,3‐dicarboxylate did not affect SRIF‐LI release. The release evoked by glutamate (100 µM) was abolished by 10 µM dizocilpine (MK‐801) plus 30 µM 1‐aminophenyl‐4‐methyl‐7,8‐methylenedioxy‐5H‐2,3‐benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK‐801 but insensitive to GYKI 52466. The AMPA‐ and KA‐evoked releases were blocked by 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) or by GYKI 52466 but were insensitive to MK‐801. The release of SRIF‐LI elicited by all four agonists was Ca2+ dependent, whereas only the NMDA‐evoked release was prevented by tetrodotoxin. Removal of Mg2+ caused increase of basal SRIF‐LI release, an effect abolished by MK‐801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.
Nicotine Effects on Dopamine Clearance in Rat Nucleus Accumbens
Authors: Hart Carl , Ksir Charles
Published: July 2010

Abstract: In vivo voltammetry was used to measure the clearance of exogenously applied dopamine (DA) in the nucleus accumbens following acute systemic nicotine administration in urethane‐anesthetized rats. The IVEC‐5 system was used for continuous in vivo electrochemical measurements. A finite amount of DA was pressure‐ejected (25–100 nl, 200 µM barrel concentration) at 5‐min intervals from micropipettes (tip diameter, 10–15 µm) positioned 250 ± 50 µm from the recording electrode. The peak DA concentration after each DA ejection was significantly decreased in rats following nicotine, but not in rats given saline. In addition, when mecamylamine was administered 20 min before nicotine it clearly antagonized nicotine effects. These results suggest that nicotine may actually facilitate DA transporter systems within the nucleus accumbens.
Thiamine, Thiamine Phosphates, and Their Metabolizing Enzymes in Human Brain
Authors: Bettendorff L. , Mastrogiacomo F. , Kish S. J. , Grisar T.
Published: July 2010

Abstract: Total thiamine (the sum of thiamine and its phosphate esters) concentrations are two‐ to fourfold lower in human brain than in the brain of other mammals. There were no differences in the total thiamine content between biopsied and autopsied human brain, except that in the latter, thiamine triphosphate was undetectable. The main thiamine phosphate‐metabolizing enzymes could be detected in autopsied brain, and the kinetic parameters were comparable to those reported in other species. Thiamine diphosphate levels were lowest in hippocampus (15 ± 4 pmol/mg of protein) and highest in mammillary bodies (24 ± 4 pmol/mg of protein). Maximal levels of thiamine and its phosphate ester were found to be present at birth. In parietal cortex and globus pallidus, mean levels of total thiamine in the oldest age group (77–103 years) were, respectively, 21 and 26% lower than those in the middle age group (40–55 years). Unlike cerebral cortex, the globus pallidus showed a sharp drop in thiamine diphosphate levels during infancy, with concentrations in the oldest group being only ∼50% of the levels present during the first 4 months of life. These data, consistent with previous observations conducted in blood, suggest a tendency toward decreased thiamine status in older people.
Effects of Chronic Ethanol Consumption on Sterol Transfer Proteins in Mouse Brain
Authors: Myers‐Payne Sean C. , Fontaine Robert N. , Loeffler Amy , Pu Lixia , Rao A. M. , Kier A. B. , Wood W. Gibson , Schroeder Friedhelm
Published: July 2010

Abstract: Although lipids are essential to brain function, almost nothing is known of lipid transfer proteins in the brain. Early reports indicates cross‐reactivity of brain proteins with antisera against two native liver sterol transfer proteins, sterol carrier protein‐2 (SCP‐2) and the liver form of fatty acid‐binding protein (L‐FABP). Herein, polyclonal antibodies raised against the recombinant liver sterol transfer proteins SCP‐2 and L‐FABP were used to identify the lipid transfer proteins in the brains of alcohol‐treated and control mice. L‐FABP was not detectable in brain of either control or chronic ethanol‐treated mice. In contrast, SCP‐2 not only was present, but its level was significantly (p < 0.05) increased 23 and 50%, respectively, in brain homogenates and synaptosomes of mice exposed to alcohol. To determine whether antibodies against the recombinant liver SCP‐2 reflected true levels of SCP‐2 in brain, the cDNA sequence for brain SCP‐2 was isolated from a brain cDNA library. The mouse brain SCP‐2 sequence was 99.99% identical to the mouse liver SCP‐2 sequence. The translated sequence differed by only one amino acid, and the replacement was conservative. Thus, unlike the fatty acid binding proteins, the SCP‐2 moieties of brain and liver are essentially identical. Polyclonal antibodies against acyl‐CoA binding protein, a lipid‐binding protein that does not bind or transfer sterol, showed that increased levels of brain SCP‐2 with chronic ethanol consumption did not represent a general increase in content of all lipid transfer proteins. Changes in the amount of SCP‐2 may contribute to membrane tolerance to ethanol.
Effects of Rumpshaker Mutation on CNS Myelin Composition and Structure
Authors: Karthigasan J. , Evans E. L. , Vouyiouklis D. A. , Inouye H. , Borenshteyn N. , Ramamurthy G. V. , Kirschner D. A.
Published: July 2010

Abstract: Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jprsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10‐kDa PLP previously reported in jprsh mice on an Rb(1.3) 1Bnr background. High‐performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jprsh myelin, further substantiating that PLP is not a component of this junctional complex. X‐raydiffraction measurements on unfixed optic nerves showed that the jprsh period is 5–10 Å larger than normal. Moreover, jprsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jprsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jprsh mutant correlate with a wider periodicity and less stable packing of the myelin.
Direct Measurement of Lipid Hydroperoxides in Iron‐Dependent Spinal Neuronal Injury
Authors: Zhang Jue‐Rong , Scherch Heidi M. , Hall Edward D.
Published: July 2010

Abstract: The relationship between iron‐dependent fetal mouse spinal cord neuron injury and the generation of endogenous lipid hydroperoxides (LOOHs) has been investigated. Cultured spinal cord neurons were incubated with ferrous iron (3–200 µM). Cell viability was measured in terms of the uptake of α‐[methyl‐3H]aminoisobutyric acid ([3H]AIB). Both endogenously and iron‐generated LOOH, i.e., free fatty acid hydroperoxide (FFAOOH), phosphatidylethanolamine hydroperoxide (PEOOH), and phosphatidylcholine hydroperoxide (PCOOH), were measured directly by an HPLC‐chemiluminescence (HPLC‐CL) assay. The FFAOOH, PEOOH, and PCOOH levels in neurons incubated with 200 µM Fe2+ for 40 min were, respectively, 22‐, 158‐, and sevenfold higher than those in non‐iron‐exposed cultures, demonstrating that phosphatidylethanolamine (PE) was most sensitive to peroxidation. The dose‐response and time course of Fe2+‐induced generation of these LOOHs were also established. In both experiments, the LOOH levels were correlated directly with loss of neuronal viability, suggesting strongly a direct relationship between lipid peroxidation and cell injury. On examination of the time course of the LOOH generation, an immediate increase in PEOOH and PCOOH levels with only 30 s of Fe2+ incubation was observed. In contrast, a lag phase in the increase in FFAOOH level (2 min after Fe2+ addition) suggested a delay in the activation of phospholipase A2 (PLA2) required for the hydrolysis and generation of FFAOOH. This culture system provides an excellent model for screening antioxidant neuroprotective compounds with regard to their ability to protect against iron‐dependent peroxidative injury and the relationship of the neuroprotection to inhibition of lipid peroxidation and/or PLA2.
Characterization and Expression of the Human A2a Adenosine Receptor Gene
Authors: Peterfreund Robert A. , MacCollin Mia , Gusella James , Fink J. Stephen
Published: July 2010

Abstract: The actions of the neurotransmitter adenosine are mediated by a family of high‐affinity, G protein‐coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans ∼9–10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5′ untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein‐coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.
Interleukin‐1β‐Mediated Regulation of μ‐Opioid Receptor mRNA in Primary Astrocyte‐Enriched Cultures
Authors: Ruzicka Bianca B. , Thompson Robert C. , Watson Stanley J. , Akil Huda
Published: July 2010

Abstract: Opioids have been found to modulate the immune system by regulating the function of immunocompetent cells. Several studies suggest that the interaction between immune and opioid systems is not unidirectional, but rather reciprocal, in nature. In the CNS, one cellular target of immune system activation is the astrocytes. These glial cells have been shown to produce the opioid peptide, proenkephalin, to express the μ‐, δ‐, and κ‐opioid receptors, and to respond to the immune factor interleukin‐1β (IL1β) with an increased proenkephalin synthesis. To characterize more completely the astrocytic opioid response to immune factor stimulation, we examined the effect of IL1β (1 ng/ml) on the μ‐receptor mRNA expression in primary astrocyte‐enriched cultures derived from rat (postnatal day 1–2) cortex, striatum, cerebellum, hippocampus, and hypothalamus. A 24‐h treatment with IL1β produced a 70–80% increase in the μ‐receptor mRNA expression in the striatal, cerebellar, and hippocampal cultures but had no effect on this expression in the cortical and hypothalamic cultures. This observation represents one of the few demonstrated increases in levels of the μ‐receptor mRNA in vitro or in vivo, since the cloning of the receptor. The enhanced μ‐receptor mRNA expression, together with the previous observation that IL1β stimulates proenkephalin synthesis in astrocytes, supports the IL1β‐mediated regulation of an astroglial opioid peptide and receptor in vitro, a phenomenon that may be significant in the modulation of the gliotic response to neuronal damage. Therefore, the astroglial opioid “system” may be important in the IL1β‐initiated, coordinated response to CNS infection, trauma, or injury.
Effect of Chronic Cocaine Treatment on μ‐ and δ‐Opioid Receptor mRNA Levels in Dopaminergically Innervated Brain Regions
Authors: Azaryan Anahit V. , Coughlin Linda J. , Búzás Beáta , Clock Barbara J. , Cox Brian M.
Published: July 2010

Abstract: The regulation of μ‐(MOR) and δ‐opioid receptor (DOR) after chronic cocaine administration has been studied. Male Sprague‐Dawley rats were treated for 3 days with saline and cocaine (50 mg/kg/day) delivered by osmotic minipump. Expression of MOR and DOR mRNA in olfactory bulb, nucleus accumbens, and caudate‐putamen (caudal and rostral parts) was estimated using quantitative competitive PCR assays after reverse transcription. No changes in the levels of mRNA for DOR were detected after exposure to cocaine in the brain regions examined. A significant increase in the level of MOR mRNA was detected in nucleus accumbens after 3 days of cocaine treatment. In caudate‐putamen and olfactory bulb, no change in MOR mRNA was observed after cocaine administration. Both SCH 23390 and eticlopride, selective antagonists of D1‐ and D2‐dopamine receptors, respectively, blocked this cocaine‐induced up‐regulation of MOR mRNA in nucleus accumbens. We suggest that endogenous opioid systems in nucleus accumbens, the brain region specifically associated with the reinforcing properties of addictive drugs, are regulated by dopaminergic mechanisms and influenced by cocaine treatment.
Cis‐Element/Cytoplasmic Protein Interaction Within the 3′‐Untranslated Region of the GLUT1 Glucose Transporter mRNA
Authors: Dwyer Kenneth J. , Boado Ruben J. , Pardridge William M.
Published: July 2010

Abstract: The posttranscriptional regulation of glucose transporter GLUT1 gene expression may be mediated by specific interactions of cytosolic proteins and regulatory cis‐elements within the untranslated regions (UTRs) of the GLUT1 mRNA. These putative cis/trans interactions were examined in the present studies with RNase T1 protection assays using 32P‐labeled GLUT1 3′‐UTR prepared from transcription plasmids and cytosolic proteins from C6 rat glioma cells. RNase T1 mapping studies localized a cis‐element to nucleotides 2,170–2,207 on the bovine GLUT1 mRNA 3′‐UTR. Ultraviolet cross‐linking of RNA/protein complexes identified two complexes having molecular masses of 88 and 44 kDa. Competition studies with synthetic RNA and oligodeoxynucleotides showed the 88‐kDa complex reacted with nucleotides 2,180–2,197 and that the 44‐kDa complex reacted with sequences within nucleotides 1,717–2,132 of the bovine GLUT1 mRNA. The GLUT1 3′‐UTR between nucleotides 2,100 and 2,300 was generated by polymerase chain reaction and subcloned at a unique Pf/MI site within the 3′‐UTR of a luciferase gene within the mammalian expression vector pGL2. Transfection of C6 rat glioma cells with the luciferase expression vector containing this portion of the GLUT1 3′‐UTR resulted in a sixfold increase in luciferase gene expression in C6 cells. The identification of these cis/trans mechanisms provides support for the hypothesis that the posttranscriptional regulation of GLUT1 gene expression may be mediated by the interaction of specific cytosolic proteins with the GLUT1 mRNA 3′‐UTR.
Effects of Somatostatin on Intracellular Calcium Concentration in PC12 Cells
Authors: Traina Giovanna , Cannistraro Salvatore , Bagnoli Paola
Published: July 2010

Abstract: Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca2+ concentration ([Ca2+]i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca2+ influx brought about by K+ depolarization. Similar effects were obtained with the application of SS analogues, such as d‐Trp8‐SS, d‐Trp8‐d‐Cys14‐SS, CGP‐23996, and SMS‐201995. In addition, treatment with cyclo‐SS, a SS antagonist, did not alter [Ca2+]i. Experiments with selective blockers of different voltage‐dependent Ca2+ channels, such as methoxyverapamil (D600) and Ω‐conotoxin GVIA, demonstrated that the effects of SS on [Ca2+]i were mediated by voltage‐dependent Ca2+ channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activity by altering Ca2+ influx.
l‐DOPA Up‐Regulates Glutathione and Protects Mesencephalic Cultures Against Oxidative Stress
Authors: Han Shan‐Kuo , Mytilineou Catherine , Cohen Gerald
Published: July 2010

Abstract: Incubation with l‐DOPA induced a rise in GSH level in cultures of fetal rat mesencephalon, mouse neuroblastoma (Neuro‐2A), human neuroblastoma (SK‐N‐MC), pig kidney epithelial cells (LLC‐PK1), and glia from newborn rat brain, but not C6 glioma cells or neuronal cultures (no glia) from the mesencephalon. The pure neuronal cultures were destroyed by incubation with l‐DOPA; added ascorbic acid or superoxide dismutase protected the cells. Washout of l‐DOPA after 48 h amplified the rise in GSH content in mixed cultures (neurons plus glia). Examination of structure‐activity relationships for elevating GSH levels in responsive cell types revealed that autooxidizable compounds (α‐methyl‐DOPA, dopamine, apomorphine, catechol, and hydroquinone) behaved similarly to l‐DOPA, whereas structural analogues that cannot undergo autooxidation (3‐O‐methyl‐DOPA, tyrosine, 2,4‐dihydroxyphenylalanine, and resorcinol) failed to elevate GSH levels. Therefore, up‐regulation of GSH appears to be a response to a mild oxidative stress. When mixed mesencephalic cultures were exposed to a strong oxidant stress by incubation with tert‐butyl hydroperoxide, a loss in viability was seen. Cultures pretreated with l‐DOPA or hydroquinone were protected from loss of viability. However, when cultures were pretreated with both l‐DOPA and ascorbate, which prevents the rise in GSH level, protection was lost, in accord with the failure to up‐regulate GSH. These results show that the up‐regulation of cellular GSH evoked by autooxidizable agents is associated with significant protection of cells. Glia play an essential role in the response of mesencephalic cell cultures. An ability to up‐regulate GSH may serve a protective role in vivo.
Lysophosphatidic Acid‐Induced Neurite Retraction in PC12 Cells: Control by Phosphoinositide‐Ca2+ Signaling and Rho
Authors: Tigyi Gabor , Fischer David J. , Sebök Ágnes , Yang Charles , Dyer David L. , Miledi Ricardo
Published: July 2010

Abstract: The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP‐ribosylation of the small‐molecular‐weight G protein Rho by the Clostridium botulinum C‐3 toxin. LPA induced a transient rise in the level of inositol 1,4,5‐trisphosphate, and retraction was blocked by inhibitors of phospholipase β. Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response. The activation of the phosphoinositide (PIP)‐Ca2+ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C—chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long‐term phorbol ester treatment—all diminished neurite retraction by interfering with LPA‐induced Ca2+ mobilization, which was required for the withdrawal of neurites. A brief 15‐min treatment with 4β‐phorbol 12‐myristate 13‐acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP‐Ca2+ second messenger system appears necessary for the Rho‐mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA‐induced morphoregulatory response.