bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐Methyl‐d‐Aspartate‐ or Glutamate‐Mediated Toxicity in Cultured Rat Cortical Neurons Is Antagonized by FPL 15896AR
Authors: Black Marsha A. , Tremblay Roger , Mealing Geoff , Ray Ranjit , Durkin Jon P. , Whitfield James F. , Blosser James , Morley Paul
Published: July 2010

Abstract: The neuroprotective action of (S)‐α‐phenyl‐2‐pyridineethanamine dihydrochloride (FPL 15896AR), a novel noncompetitive N‐methyl‐d‐aspartate (NMDA) receptor antagonist, was examined in primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (50 µM) or glutamate (50 µM) for 15 min resulted in the death of 85–95% of the neurons during the next 24 h. This neurotoxicity was completely eliminated by adding FPL 15896AR (50 µM) to the cultures during the time of NMDA or glutamate exposure. Neuroprotective concentrations of FPL 15896AR also inhibited other acute effects of NMDA. FPL 15896AR (50 µM) prevented the loss of membrane‐associated protein kinase C activity that developed by 4 h after transient exposure to 50 µM NMDA or 50 µM glutamate. FPL 15896AR also reduced by ∼35% the magnitude of NMDA‐triggered increases in intracellular free Ca2+ concentration in the cortical cultures. These data indicate that NMDA‐mediated toxicity in cultured cortical neurons can be blocked by the NMDA antagonist FPL 15896AR.
Thiamine Deficiency‐Induced Partial Necrosis and Mitochondrial Uncoupling in Neuroblastoma Cells Are Rapidly Reversed by Addition of Thiamine
Authors: Bettendorff L. , Sluse F. , Goessens G. , Wins P. , Grisar T.
Published: July 2010

Abstract: Culture of neuroblastoma cells in a medium of low‐thiamine concentration (6 nM) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate‐dependent α‐ketoglutarate dehydrogenase activity is impaired. When thiamine (10 µM) is added to the cells, the O2 consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate‐independent succinate dehydrogenase, is also able to restore a normal O2 consumption (with respiratory control) in digitonin‐permeabilized thiamine‐deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernicke's encephalopathy.
Effects of Prostaglandin E2 and Capsaicin on Behavior and Cerebrospinal Fluid Amino Acid Concentrations of Unanesthetized Rats: A Microdialysis Study
Authors: Malmberg Annika B. , Hamberger Anders , Hedner Thomas
Published: July 2010

Abstract: Prostaglandin E2 (PGE2) delivered to the spinal cord produces an increased sensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimuli. The mechanisms that underlie this effect remain unknown, but a PGE2‐evoked enhancement of spinal neurotransmitter release may be involved. To address this hypothesis, we examined the effect of PGE2 on CSF concentrations of amino acids and also the modulatory effect of PGE2 on capsaicin‐evoked changes of spinal amino acid concentrations using a microdialysis probe placed in the lumbar subarachnoid space. Amino acids were quantified using HPLC with fluorescence detection. Addition of 1 mM, but not 10 or 100 µM, PGE2 to the perfusate for a 10‐min period (flow rate, 5 µl/min) evoked an immediate increase (80–100%) in glutamate (Glu), aspartate (Asp), taurine (Tau), glycine (Gly), and γ‐aminobutyric acid (GABA) concentrations. Similarly, capsaicin infusion (0.1–10 µM) induced a dose‐dependent increase in Glu, Asp, Tau, Gly, GABA, and ethanolamine levels. Significant increases in amino acid levels evoked by PGE2 or capsaicin were associated with a touch‐evoked allodynia. The combination of PGE2 (10 µM) and capsaicin (0.1 or 1.0 µM) at concentrations that individually had no effect together evoked a significant increase (60–100%) in Glu, Asp, Tau, Gly, and GABA concentrations and produced tactile allodynia. These data demonstrate that spinally delivered PGE2 or capsaicin substantially elevates CSF concentrations of both excitatory and inhibitory amino acids. The capacity of PGE2 to enhance and prolong capsaicin‐evoked amino acid concentrations may be one of the mechanisms by which spinal PGE2 produces hyperalgesia and allodynia.
Brain Quinolinic Acid in Chronic Experimental Hepatic Encephalopathy: Effects of an Exogenous Ammonium Acetate Challenge
Authors: Bergqvist Peter B. F. , Heyes Melvyn P. , Bugge Mogens , Bengtsson Finn
Published: July 2010

Abstract: Elevated brain concentrations of the neurotoxin and NMDA receptor agonist quinolinic acid (QUIN) have been demonstrated in portacaval‐shunted (PCS) rats, a chronic hepatic encephalopathy (HE) model. Increased brain QUIN levels have also been shown in acute hyperammonemic rats. In the present study, the plasma and brain (neocortical) QUIN levels in chronic PCS rats were investigated. The study also included a single exogenous ammonium acetate (NH4Ac; 5.2 mmol/kg, i.p.) challenge to precipitate a reversible hepatic coma. Compared with sham‐operated controls, chronic PCS rats exhibited decreased rather than increased plasma and brain QUIN levels. The plasma‐to‐brain QUIN ratio was not found to be altered. The NH4Ac administration induced coma in all of the PCS rats 20–25 min after the challenge, and this coma was resolved within 60–75 min. No relevant temporal relationship between changes in brain QUIN levels and the neurological status in the PCS rats was observed. Therefore, our results do not support the contention that increased brain QUIN levels per se are involved in the pathogenesis of HE.
Characterization of TrkB Receptor‐Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival
Authors: Zirrgiebel Ute , Ohga Yoko , Carter Bruce , Berninger Benedikt , Inagaki Naoyuki , Thoenen Hans , Lindholm Dan
Published: July 2010

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5). Here, we report that both truncated and full‐length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT‐4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT‐4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C‐γ (PLC‐γ) and SH2‐containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC‐γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival‐promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c‐ras in a concentration‐dependent manner. These results suggest that two different pathways, the c‐ras and the PLC‐γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.
Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity
Authors: Pavlaković Goran , Kane Michael D. , Eyer Charles L. , Kanthasamy Anumantha , Isom Gary E.
Published: July 2010

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µM TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24‐h period. TMT treatment was accompanied by a low level of PKC down‐regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µM). Phorbol myristate‐induced PKC down‐regulation or inhibition with chelerythrine provided protection against TMT‐induced cytotoxicity. It was concluded that TMT‐induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.
Characterization of the Thyroid Hormone Transport System of Cerebrocortical Rat Neurons in Primary Culture
Authors: Chantoux Françoise , Blondeau Jean‐Paul , Francon Jacques
Published: July 2010

Abstract: The uptake of 3′,3,5‐triiodo‐l‐thyronine (T3) and l‐thyroxine (T4) by primary cultures derived from rat brain hemispheres was studied under initial velocity conditions, at 25°C. Uptake of both hormones was carrier mediated and obeyed simple Michaelis‐Menten kinetics. The Km of T3 uptake was very similar to that of T4, and did not vary significantly from day 1 to 4 in culture (310–400 nM). The maximal velocity (Vmax) of T3 uptake nearly doubled between day 1 and 4 of culture (41 ± 3 vs. 70 ± 5 pmol/min/mg of DNA, respectively). The Vmax of T4 uptake did not change (28 ± 8 and 31 ± 4 pmol/min/mg of DNA on days 1 and 4, respectively). The rank order of unlabeled thyroid hormone analogues to compete with labeled T3 or T4 uptakes were the same (T3 > T4 > 3′,5′,3‐triiodo‐l‐thyronine > 3′,3,5‐triiodo‐d‐thyronine > triiodothyroacetic acid), indicating that the transport system is stereospecific. Unlabeled T4 was a stronger competitor of labeled T4 uptake than of labeled T3 uptake, whereas unlabeled T3 had the same potency for both processes. These results suggest that T3 and T4 are transported either by two distinct carriers or by the same carrier bearing separate binding sites for each hormone. They also indicate that the efficiency of T3 uptake increases during neuronal maturation.
Effects of Dibutyryl Cyclic AMP and Retinoic Acid on the Differentiation of Dopamine Neurons: Prevention of Cell Death by Dibutyryl Cyclic AMP
Authors: Mena Maria Angeles , Casarejos Maria José , Bonin Ana , Ramos Jose Antonio , De Yébenes Justo García
Published: July 2010

Abstract: Immature neurons, including fetal and tumoral cells, are used for investigating neuronal differentiation in vitro. The human neuroblastoma cell line NB69 could be induced to differentiate to dopamine or acetylcholine neurons by different compounds, including neurotrophins and activators of the protein kinases. In these NB69 cells dibutyryl cyclic AMP (dbcAMP) at 2 mM reduced the division rate and increased the levels of catecholamines, tyrosine hydroxylase (TH) activity, and monoamine oxidase activity. The dbcAMP also increased cell size, dendritic arborization, density of the sites for high‐affinity dopamine uptake, and activity of choline acetyltransferase. In fetal rat midbrain neurons treatment with dbcAMP increased the levels of dopamine and the number of TH‐immunoreactive neurons in the culture. When embryonic day 14 fetal midbrain neurons, previously exposed to 1 µM retinoic acid (a compound that severely reduces the number of fetal midbrain dopamine neurons), were treated with dbcAMP, the levels of dopamine and the number of TH‐immunoreactive cells returned to normal levels. This suggests that dbcAMP induces the differentiation to dopamine neurons of quiescent progenitor or facilitates expression of the dopamine phenotype in immature neurons. Therefore, dbcAMP not only differentiates uncommitted immature dopamine neurons, but also reverses the antidopaminergic effects of retinoic acid. These properties of dbcAMP could be of therapeutic value in Parkinson's disease.
3.6 kb of the 5′ Flanking DNA Activates the Mouse Tyrosine Hydroxylase Gene Promoter Without Catecholaminergic‐Specific Expression
Authors: Morgan William W. , Walter Christi A. , Windle Jolene J. , Sharp Z. Dave
Published: July 2010

Abstract: The tyrosine hydroxylase (TH) gene is expressed exclusively in cells and neurons that synthesize and release l‐DOPA or catecholamines. To further understand the molecular genetic mechanisms that regulate this cell‐type specific expression, a chimeric gene was prepared by linking 3.6 kb of the 5′ flanking DNA of the mouse TH gene, including the +1 initiation site for transcription, to an E. coliβ‐galactosidase reporter. This fusion gene (TH3.6LAC) was used to prepare transgenic mice, and the tissue distribution of expression of TH3.6LAC was determined by the measurement of β‐galactosidase enzymatic activity and/or by the detection of the transcription product of the chimeric gene by RNase protection assays. In two separate founder lines, TH3.6LAC expression was observed in every region of the brain that was examined, including the olfactory bulb, brainstem, cerebellum, diencephalon, hippocampus, striatum, and cerebral cortex. Expression of TH3.6LAC was observed in the adrenal gland of one founder line but not in the other. TH3.6LAC activation was undetectable in peripheral organs that were examined, including the liver, heart, salivary gland, kidney, lung, and spleen. Although 3.6 kb of the 5′ regulatory DNA of the mouse TH gene is sufficient to activate the TH fusion gene in the mouse, it is not enough to restrict its expression to catecholaminergic cells.
Heterogeneity in the Expression Pattern of Two Ganglioside Synthase Genes During Mouse Brain Development
Authors: Yamamoto Akihito , Haraguchi Masashi , Yamashiro Shuji , Fukumoto Satoshi , Furukawa Keiko , Takamiya Kogo , Atsuta Mitsuru , Shiku Hiroshi , Furukawa Koichi
Published: July 2010

Abstract: Gangliosides are synthesized by sequential catalytic reaction of multiple glycosyltransferases. GM2/GD2 synthase and GD3 synthase are key enzymes for ganglioside synthesis, because their relative activities regulate the main profiles of ganglioside expression. Mouse GD3 synthase (EC 2.4.99.8) cDNA was cloned by eukaryotic expression cloning, and its mRNA expression as well as that of GM2/GD2 synthase gene during the development of the mouse CNS was analyzed by using northern blotting, reverse transcription‐polymerase chain reaction, and in situ hybridization. When brain tissue was analyzed as a whole mass, a typical pattern corresponding to the reported findings obtained by biochemical analyses was observed, i.e., high expression of GD3 synthase gene in the early stage and gradual increase of GM2/GD2 synthase gene expression in the late stage of the development. However, the results of in situ hybridization of these two genes revealed that the expression kinetics of these two genes were heterogeneous among various sites in the brain under development. These findings suggest that various expression patterns of the two genes reflect differences in the course of the development of individual sites, and also different ganglioside components are required in individual portions of the brain for development and maintenance of the function.
Identification of Evolutionary Conserved Regulatory Sequences in the 5′ Untranscribed Region of the Neural‐Specific Ubiquitin C‐Terminal Hydrolase (PGP9.5) Gene
Authors: Mann Derek A. , Trowern Angus R. , Lavender F. Louise , Whittaker Paul A. , Thompson Rod J.
Published: July 2010

Abstract: The structure at the 5′ end of the gene encoding neural‐specific protein gene product 9.5 (PGP9.5) has been compared between two evolutionary distant species: the human and Monodelphis domestica. In contrast to the highly conserved coding sequences of the gene, only a 48% identity was found across a 1‐kb stretch of 5′ untranslated and untranscribed DNA. Promoter function studies performed on the human sequence identified a 233‐bp CpG‐rich minimal promoter. Truncation mutagenesis revealed the presence of essential positive cis‐acting regulatory sequences within the region −182 to −123 relative to the transcription initiation site. Sequence alignment analysis of the human and Monodelphis promoter sequences showed 76% identity in this 59‐bp region of the gene. A perfectly conserved 12‐bp sequence (PSN) located within this region acts as a non‐cell‐specific activator of transcription in a heterologous reporter gene (pBLCAT2). PGP9.5 gene expression can be readily detected in human neuroblastoma cell lines but is absent in nonneuronal cell lines such as HeLa. Studies on the cell type specificity of the human PGP9.5 promoter demonstrated that in contrast to the endogenous gene, the promoter is active in HeLa cells. However, the promoter displays higher levels of activity in human neuroblastoma cell lines. A conserved 16‐bp sequence located at −356 (motif 5) was able to reduce the activity of a heterologous minimal promoter specifically in HeLa cells. In conclusion, we have shown that expression of the PGP9.5 gene is regulated by evolutionary conserved positive and negative cis‐acting sequences located in the untranscribed region of the gene.
Stimulation of Cloned Human Serotonin 5‐HT1Dβ Receptor Sites in Stably Transfected C6 Glial Cells Promotes Cell Growth
Authors: Pauwels Petrus J. , Wurch Thierry , Amoureux Marie‐Claude , Palmier Christiane , Colpaert Francis C.
Published: July 2010

Abstract: The involvement of serotonin 5‐HT1Dβ receptor sites was investigated in the growth of rat C6 glial cells permanently transfected with a gene encoding a human 5‐HT1Dβ receptor. The 5‐HT receptor identity of control and transfected C6 glial/5‐HT1Dβ cells was determined by reverse transcription‐polymerase chain reaction using primers specific for rat 5‐HT1A, rat 5‐HT1B, rat 5‐HT1Dα, human 5‐HT1Dβ, and rat 5‐HT2A receptor genes. Constitutive mRNA for 5‐HT2A receptors was present in control and transfected C6 glial/5‐HT1Dβ cells, whereas mRNA for 5‐HT1Dβ receptor sites was only present in the transfected C6 glial/5‐HT1Dβ cell line. 5‐HT inhibited forskolin‐stimulated cyclic AMP formation and promoted cell growth, in contrast to the absence of any measurable effect in pcDNA3 plasmid‐transfected and nontransfected C6 glial cells. The 5‐HT effects could be mimicked by sumatriptan (EC50 = 44–76 nM) and were totally and partially blocked by methiothepin (IC50 = 9 nM) and GR 127,935 {2′‐methyl‐4′‐(5‐methyl[1,2,4]oxadiazol‐3‐yl)‐biphenyl‐4‐carboxylic acid [4‐methoxy‐3‐(4‐methylpiperazin‐1‐yl)phenyl]amide; IC50 = 97 pM}, respectively. No effect on cell growth was measured with the 5‐HT2 receptor agonist DOI [1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane; 10 µM], suggesting that 5‐HT2A receptors are not involved in the 5‐HT‐stimulated C6 glial/5‐HT1Dβ cell growth. Dibutyryl‐cyclic AMP (0.3 mM)‐treated cultures did not show sumatriptan‐promoted cell growth, indicating an inhibitory role for cyclic AMP in the cell growth mediated by 5‐HT1Dβ receptor sites. In conclusion, 5‐HT promotes cell proliferation in transfected C6 glial/5‐HT1Dβ cells by stimulation of 5‐HT1Dβ receptor sites. These receptor sites may be a new target to modulate the growth of tumor cells.
Glutamate Induces a Calcineurin‐Mediated Dephosphorylation of Na+,K+‐ATPase that Results in Its Activation in Cerebellar Neurons in Culture
Authors: Marcaida Goizane , Kosenko Elena , Miñana María‐Dolores , Grisolía Santiago , Felipo Vicente
Published: July 2010

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca2+ and Na+ into the neuron. To maintain Na+ homeostasis, the excess Na+ entering through the ion channel should be removed by Na+,K+‐ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na+,K+‐ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate‐induced activation of Na+,K+‐ATPase was dose dependent: It was appreciable (37%) at 0.1 µM and peaked (85%) at 100 µM. The increase in Na+,K+‐ATPase activity by glutamate was prevented by MK‐801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12‐myristate 13‐acetate, an activator of protein kinase C, indicating that activation of Na+,K+‐ATPase is due to decreased phosphorylation by protein kinase C. W‐7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na+,K+‐ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na+,K+‐ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na+,K+‐ATPase.
Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN‐62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry
Authors: Maurer Jennifer A. , Wenger Bryan W. , McKay Dennis B.
Published: July 2010

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K‐252a, H‐7, and H‐8) and reportedly selective protein kinase inhibitors (KN‐62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and 45Ca2+ uptake. Treatment of chromaffin cells with 10 µM K‐252a, 50 µM H‐7, or 50 µM H‐8 induced changes in cell morphology within 3 h; these compounds also induced a time‐dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca2+/phospholipid‐dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN‐62, a selective inhibitor of Ca2+/calmodulin‐dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC50 value of 0.32 µM), but had no effect on cell morphology. The reduction of secretory function induced by 1 µM KN‐62 was significant within 5 min and rapidly reversible. Unlike H‐7, H‐8, and staurosporine, KN‐62 significantly inhibited stimulated 45Ca2+ uptake. KN‐04, a structural analogue of KN‐62 that does not inhibit CaMK II, inhibited stimulated 45Ca2+ uptake and catecholamine release like KN‐62. These studies indicate that KN‐62 inhibits secretory function via the direct blockade of activated Ca2+ influx. The nonselective inhibitors, K‐252a, H‐7, H‐8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.
Overexpression of Alternative Human Acetylcholinesterase Forms Modulates Process Extensions in Cultured Glioma Cells
Authors: Karpel Rachel , Sternfeld Meira , Ginzberg Dalia , Guhl Eva , Graessmann Adolf , Soreq Hermona
Published: July 2010

Abstract: In addition to its well‐known synaptic function, acetylcholinesterase was recently shown to stimulate neurite outgrowth from cultured chick neurons in a manner unrelated to its catalytic activity. It remained unclear, however, whether each of the variant acetylcholinesterase enzyme forms can promote such process extension and whether this effect of acetylcholinesterase was limited to neurite outgrowth. Using DNA microinjections and stable transfections of cultured glioma cells, we explored the possibility that specific acetylcholinesterase isoforms affect cellular development and morphology of CNS astrocytes. Cells microinjected with human ACHEDNA constructs that differ in their exon‐intron composition displayed rapid yet stable induction of cell body enlargement and process extensions. Cells transfected with ACHEDNA carrying the neuronal‐characteristic 3′‐E6 domain also displayed stable process extensions. However, stable transfections with ACHEDNAs including the 3′‐alternative I4/E5 region induced the appearance of small, round cells in a dominant manner. This was associated with expression of I4/E5‐ACHEmRNA transcripts and the production of soluble acetylcholinesterase monomers that were catalytically indistinguishable from the 3′‐E6 enzyme but displayed higher electrophoretic mobility than that of the 3′‐E6 form. Thus, variable expression levels and alternative splicing modes of the ACHE gene correlated in these experiments with glial development in a manner that was apparently unrelated to catalysis.
GABAA Receptors Modulate Early Spontaneous Excitatory Activity in Differentiating P19 Neurons
Authors: Lin Pengxin , Kusano Kiyoshi , Zhang Qianlin , Felder Christian C. , Geiger Peter M. , Mahan Lawrence C.
Published: July 2010

Abstract: P19 embryonic carcinoma (EC) stem cells are pluripotent and are efficiently induced to differentiate into neurons and glia with retinoic acid (RA) treatment. Within 5 days, a substantial number of differentiating P19 cells express gene products that are characteristic of a neuronal phenotype. P19 neurons were used as a model to explore the relationship between neuronal “differentiation” in vitro and the acquisition of γ‐aminobutyric acid (GABAA) receptors and functional GABA responses. Pulse‐labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3–4 days after treatment with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA‐1. Subsequently, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5–10 DIV after RA, a significant number of neurons (∼80–90%) expressed immunocytochemically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenotypic markers were maintained for >30 DIV after RA. Under current‐clamp conditions, random, low‐amplitude, spontaneous electrical activity appeared in neurons within the first few days after RA treatment and this was blocked by the specific GABAA receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that were similarly attenuated by bicuculline. In neurons > 5 DIV after RA, exogenous application of GABA elicited similar action potentials. The onset of excitatory responses to GABA or muscimol in voltage‐clamped neurons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura‐2 imaging studies, the exogenous application of GABA resulted in neuron‐specific increases in intracellular Ca2+. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of functional GABAA receptors.
Assessment of Amyloid β Protein in Cerebrospinal Fluid as an Aid in the Diagnosis of Alzheimer's Disease
Authors: Southwick Paula C. , Yamagata Susan K. , Echols Charles L. , Higson Gail J. , Neynaber Scott A. , Parson Robert E. , Munroe William A.
Published: July 2010

Abstract: The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39–42‐amino‐acid protein, amyloid β protein (Aβ). This study examined whether the measurement of Aβ levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non‐AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and Aβ concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean Aβ value for the AD group (15.9 ± 6.8 ng/ml) was not significantly different from that for the non‐AD control group (13.0 ± 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. Aβ values did not correlate with age (r = −0.05, p = 0.59), severity of cognitive impairment (r = 0.22, p = 0.21), or duration of AD symptoms (r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of Aβ in CSF. Aβ immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of Aβ reactivity. In conclusion, the total CSF Aβ level is not a useful marker for current diagnosis of AD.
Tissue Distribution and Immunocytochemical Localization of Neurotrophin‐3 in the Brain and Peripheral Tissues of Rats
Authors: Katoh‐Semba Ritsuko , Kaisho Yoshihiko , Shintani Asae , Nagahama Masato , Kato Kanefusa
Published: July 2010

Abstract: The tissue distribution of neurotrophin‐3 (NT‐3) was investigated in rats at 1 month of age using a newly established, sensitive two‐site enzyme immunoassay system for NT‐3, as well as the immunocytochemical localization of this protein. The immunoassay for NT‐3 enabled us to quantify NT‐3 at levels > 3 pg per assay. In the rat brain, NT‐3 was detectable only in the olfactory bulb (0.54 ng/g wet weight), cerebellum (0.71 ng/g), septum (0.91 ng/g), and hippocampus (6.3 ng/g). By contrast, NT‐3 was widely distributed in peripheral tissues. Appreciable levels of NT‐3 were also found in the thymus (31 ng/g), heart (38 ng/g), diaphragm (21 ng/g), liver (45 ng/g), pancreas (892 ng/g), spleen (133 ng/g), kidney (40 ng/g), and adrenal gland (46 ng/g). An antibody specific for NT‐3 bound to pyramidal cells in the CA2‐CA4 regions of the hippocampus, to A cells in the islets of Langerhans in the pancreas, to unidentified cells in the red pulp of the spleen, to liver cells, and to muscle fibers in the diaphragm from rats at 1 month of age. Molecular masses of NT‐3‐immunoreactive proteins in the hippocampus and pancreas were 14 and 12 kDa, respectively. Thus, in rats, NT‐3 was detected in restricted regions of the brain and in the visceral targets of the nodose ganglia at high concentrations. Our present results suggest that NT‐3 not only functions as a classical target‐derived neurotrophic factor but also can play other roles.
Investigation of the Role of Conserved Serine Residues in the Long Form of the Rat D2 Dopamine Receptor Using Site‐Directed Mutagenesis
Authors: Woodward R. , Coley C. , Daniell S. , Naylor L. H. , Strange P. G.
Published: July 2010

Abstract: Three serine residues (Ser193, Ser194, Ser197) in the fifth transmembrane‐spanning region of the D2 dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand‐binding experiments with [3H]spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 µM). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist‐binding curves in the absence of GTP, when higher and lower affinity agonist‐binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala193 and Ala194 mutations, but the Ala197 mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.
Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament‐Associated Kinase and by Casein Kinase I
Authors: Hollander Brian A. , Bennett Gudrun S. , Shaw Gerry
Published: July 2010

Abstract: We have shown previously that a neurofilament (NF)‐associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF‐M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI‐7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two‐dimensional phosphopeptide maps of NF‐M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross‐reactivity of NFAK with an antibody raised against CKI. We have also identified Ser502, Ser528, and Ser536 as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF‐M sequences. Neither Ser502 nor Ser536 has been identified previously as NF‐M phosphorylation sites.
Increased Brain Levels of Platelet‐Activating Factor in a Murine Acquired Immune Deficiency Syndrome Are NMDA Receptor‐Mediated
Authors: Nishida Keiji , Markey S. P. , Kustova Yelena , Morse Herbert C. , Skolnick Phil , Basile Anthony S. , Sei Yoshitatsu
Published: July 2010

Abstract: Mice infected with the LP‐BM5 murine leukemia virus (MuLV) develop an immunodeficiency syndrome (murine AIDS) and an encephalopathy characterized by impaired spatial learning and memory. Because platelet‐activating factor (PAF) has been implicated in the pathogenesis of HIV‐associated dementia complex, brain PAF levels were measured in LP‐BM5 MuLV‐infected mice. PAF levels in cerebral cortex and hippocampus were significantly increased at 6 and 12 weeks after LP‐BM5 MuLV inoculation, whereas significant increases in striatal and cerebellar PAF levels were observed only at 12 weeks after inoculation. Administration of the NMDA antagonist MK‐801 significantly reduced the increased PAF levels in the cerebral cortex and hippocampus of LP‐BM5 MuLV‐infected mice. These results indicate that the LP‐BM5 MuLV‐induced increases in brain PAF levels are the result of NMDA receptor activation and are consistent with the hypothesis that elevated CNS PAF levels contribute to the behavioral deficits observed in LP‐BM5 MuLV‐infected mice.
Coupling of ETB Endothelin Receptor to Mitogen‐Activated Protein Kinase Stimulation and DNA Synthesis in Primary Cultures of Rat Astrocytes
Authors: Lazarini F. , Strosberg A. D. , Couraud P. O. , Cazaubon S. M.
Published: July 2010

Abstract: Astrocytes have been shown to express endothelin (ET) receptors functionally coupled, via different heterotrimeric G proteins, to several intracellular pathways. To assess the relative contribution of each subtype in the astrocytic responses to ET‐1, effects of BQ123, an antagonist selective for the ET receptor subtype A (ETA‐R), and IRL1620, an agonist selective for the ET receptor subtype B (ETB‐R), were investigated in primary cultures of rat astrocytes. Binding experiments indicated that the ETB‐R is the predominant subtype in these cells. Inhibition of forskolin‐stimulated cyclic AMP production was observed under ETB‐R stimulation. Bordetella pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway is coupled to the ETB‐R via Gi protein. Increases of tyrosine phosphorylation of cellular proteins, stimulation of mitogen‐activated protein kinase (MAPK), and DNA synthesis were also found to be mediated by the ETB‐R, but through PTX‐insensitive G protein. IRL1620‐induced MAPK activation involved the adapter proteins Shc and Grb2 and the serine/threonine kinase Raf‐1. This study reveals that the various effects of ET‐1 in astrocytes are mediated by the ETB‐R, which couples to multiple signaling pathways including the MAPK cascade.
Characterization of the Effects of Lithium and Inositol on Phosphoinositide Turnover in Cerebellar Granule Cells in Primary Culture
Authors: Del Río E. , Nicholls D. G. , Downes C. P.
Published: July 2010

Abstract: The effect of lithium on inositol phospholipid resynthesis in primary cultures of cerebellar granule cells was studied. During activation of phospholipase C by the combined action of a muscarinic agonist and mild depolarization, the levels of inositol phospholipids as well as the inositol phospholipid precursor CMP‐phosphatidate appeared highly sensitive to lithium with half‐maximal accumulation of CMP‐phosphatidate attained at 0.5 mM LiCl, a concentration close to that in the plasma of patients subjected to lithium therapy. Under the same conditions, the effect of lithium on inositol phospholipid metabolism appeared to be mediated by depletion of cytoplasmic free inositol content. This was indicated by the observation that preincubation for 48 h in high extracellular inositol concentrations could decrease or delay the depletion of inositol phospholipids and the accumulation of CMP‐phosphatidate induced by 10 mM LiCl. Because even relatively high concentrations of extracellular inositol (500 µM) only partially prevented inositol phospholipid depletion, cerebellar granule cells appear to have a comparatively low capacity to accumulate inositol intracellularly, in comparison with other brain cells in culture. The relationship between CMP‐phosphatidate accumulation and phospholipase C activity has also been investigated using a range of agonists that have been reported to act on cerebellar granule cells.
Extracellular Dopamine, Norepinephrine, and Serotonin in the Nucleus Accumbens of Freely Moving Rats During Intracerebral Dialysis with Cocaine and Other Monoamine Uptake Blockers
Authors: Li Ming‐Ya , Yan Qing‐Shan , Coffey Lori L. , Reith Maarten E. A.
Published: July 2010

Abstract: Monoamine‐uptake blockers were applied focally (0.1–1,000 µM) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine‐uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin‐uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine‐uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.
Antagonism of Serotonin 5‐HT1A Receptors Potentiates the Increases in Extracellular Monoamines Induced by Duloxetine in Rat Hypothalamus
Authors: Engleman Eric A. , Robertson David W. , Thompson Dennis C. , Perry Kenneth W. , Wong David T.
Published: July 2010

Abstract: In the current study we examined the effects of coadministration of a serotonin 5‐HT1A antagonist, (±)‐1‐(1H‐indol‐4‐yloxy)‐3‐(cyclohexylamino)‐2‐propanol maleate (LY 206130), and a dual 5‐HT and norepinephrine (NE) uptake inhibitor, duloxetine, on extracellular levels of NE, 5‐HT, dopamine (DA), 5‐hydroxyindoleacetic acid, and 3,4‐dihydroxyphenylacetic acid in rat hypothalamus microdialysates. LY 206130 (3.0 mg/kg, s.c.) alone significantly increased NE and DA levels by 60 and 34%, respectively, without affecting 5‐HT levels. Duloxetine administration at 4.0 mg/kg, i.p. alone produced no significant changes in levels of 5‐HT, NE, or DA. In contrast, when LY 206130 and duloxetine were coadministered at 3.0 mg/kg, s.c. and 4.0 mg/kg, i.p., respectively, 5‐HT, NE, and DA levels increased to 5.7‐, 4.8‐, and threefold over their respective basal levels. These data demonstrate that antagonism of somatodendritic 5‐HT1A autoreceptors and concomitant inhibition of 5‐HT and NE uptake with duloxetine may promote synergistic increases in levels of extracellular 5‐HT, NE, and DA in hypothalamus of conscious, freely moving rats.
l‐3,4‐Dihydroxyphenylalanine Increases the Quantal Size of Exocytotic Dopamine Release In Vitro
Authors: Pothos Emmanuel , Desmond Mark , Sulzer David
Published: July 2010

Abstract: The catecholamine precursor l‐3,4‐dihydroxyphenylalanine (l‐DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of l‐DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 mM potassium‐containing media, l‐DOPA (100 µM for 1 h) increased DA release by >10‐fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine‐uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, l‐DOPA (50 µM for 1 h) elevated DA release in high‐potassium media by 370%. Amperometric measurements demonstrated that l‐DOPA (50 µM for 40–70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that l‐DOPA can increase stimulation‐dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size.
The Latency of Exocytosis Varies with the Mechanism of Stimulated Release in PC12 Cells
Authors: Zerby Susan E. , Ewing Andrew G.
Published: July 2010

Abstract: To compare the time course of different mechanisms of chemically stimulated release, amperometric detection of dopamine was carried out at single PC12 cells. The rapid response of carbon fiber microelectrodes allowed the detection of single exocytotic events, thus providing time‐resolved information about the dynamics of stimulated release, in particular the latency between the stimulation of a cell and the secretion of catecholamines. On rapid depolarization of the cell membrane caused by application of 105 mM K+, almost immediate (6 ± 1 s) release of dopamine was observed. Stimulation with 1 mM nicotine, involving the stimulant binding to a ligand‐gated ion channel, resulted in a short (37 ± 5 s) delay between stimulation and secretion. Application of 1 mM muscarine to the cells caused a long (103 ± 11 s) latency before exocytosis was detected. A biphasic response that appeared to be similar to a combination of nicotine‐ and muscarine‐stimulated release was observed when cells were stimulated with 10 mM acetylcholine. Thus, it appears that the dynamics of stimulated release at single PC12 cells is significantly affected by the mechanism leading to exocytosis.
Time Dependence of N‐Acetyl‐Aspartate, Lactate, and Pyruvate Concentrations Following Spinal Cord Injury
Authors: Falconer J. C. , Liu S.‐J. , Abbe R. A. , Narayana P. A.
Published: July 2010

Abstract: The time dependence of N‐acetyl‐aspartate (NAA) concentrations relative to lactate and pyruvate in the injured rat spinal cord was investigated. Segments of spinal cord from regions rostral, caudal, and at the epicenter of the injury were analyzed. NAA concentrations were determined by gas chromatography‐mass spectrometry and lactate and pyruvate concentrations were determined by UV spectroscopy at 20 min, 60 min, 2 h, 8 h, 24 h, 3 days, and 1 week after injury. NAA levels fell most significantly at the epicenter of the injury, reaching 30% of basal levels within 24 h. In all segments, lactate levels increased significantly shortly after injury, peaking at two to five times normal basal levels between 20 and 60 min after injury. Rostral and caudal to the injury site, lactate elevations and NAA reductions were less dramatic. Pyruvate concentrations were not significantly altered in any of the sections after injury. The temporal and spatial relationships of NAA and lactate changes indicated that ischemic conditions due to injury in the upper thoracic rat spinal cord were distributed asymmetrically. Acute ischemia was more severe caudal to the injury site, and NAA concentrations were more severely impaired in the rostral direction. The results suggest that the extent of neuronal degeneration due to spinal cord injury does not correlate directly with acute ischemic severity as measured by the lactate/pyruvate ratio, and may be more closely related to secondary changes in the neuronal environment.
Identification of a Neuropeptide and Neuropeptide‐Processing Enzymes in Aqueous Humor Confers Neuroendocrine Features to the Human Ocular Ciliary Epithelium
Authors: Ortego Javier , Escribano Julio , Crabb John , Coca‐Prados Miguel
Published: July 2010

Abstract: The ocular ciliary epithelium, the site of aqueous humor secretion in the mammalian eye, is believed to play a key function in signaling mechanisms that regulate the rate of secretion, and thus intraocular pressure. One possible way of mediating these signaling functions is through neuropeptides and hormones secreted into the aqueous humor and acting on target tissues. We recently identified a cDNA clone sharing 100% identity with carboxypeptidase E (CPE), a neuropeptide‐processing enzyme. Utilizing polymerase chain reaction, we further identified and characterized another processing enzyme, the peptidylglycine α‐amidating monooxygenase (PAM), and the neuropeptide secretogranin II, a molecular marker restricted to neuroendocrine tissues. Using specific probes, we found that the nonpigmented ciliary epithelial cells express CPE, PAM, and secretogranin II mRNA, and protein. We also found that CPE and secretogranin II are abundant in aqueous humor. Treatment of cultured ciliary epithelial cells with veratridine and phorbol ester up‐regulates CPE and PAM. Secretogranin II was found to be induced by veratridine, whereas phorbol ester had little effect, suggesting different mechanisms for secretion. The results demonstrate that secretogranin II, CPE, and PAM represent a specialized group of neuropeptide and neuropeptide‐processing enzymes secreted by the ciliary epithelial cells which may confer to them neuroendocrine functions in cell‐cell communication or cell signaling.
A Subtype of κ‐Opioid Receptor Mediates Inhibition of High‐Affinity GTPase Inherent in Gi1 in Guinea Pig Cerebellar Membranes
Authors: Ueda Hiroshi , Misawa Hidemi , Fukushima Nobuyuki , Katada Toshiaki , Ui Michio , Satoh Masamichi
Published: July 2010

Abstract: The κ‐opioid receptor agonists including U‐50,488H and dynorphin A (1–17) in ranges of 0.1–100 nM inhibited the hydrolysis of GTP to GDP (Pi release) inherent in GTP‐binding proteins (G proteins) in guinea pig cerebellar membranes. U‐50,488H inhibited only high‐affinity GTPase activity, not low‐affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 µM. The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 µg/ml but not with preactivated cholera toxin (30 µg/ml). Similar blockade of κ‐receptor‐mediated inhibition was also observed when membranes were pretreated with a low concentration (8 µM) of N‐ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP‐ribosylated by PTX; but this treatment caused no significant change in κ‐agonist binding. When purified Gi1, but not Go, was reconstituted into membranes pretreated with NEM, the κ‐receptor‐mediated inhibition was recovered. These findings suggest that a subtype of κ‐opioid receptor is coupled to inhibition of intrinsic activity of Gi1.