bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct Comparison of the Response of Voltammetry and Microdialysis to Electrically Evoked Release of Striatal Dopamine
Authors: Lu Yi , Peters Jennifer L. , Michael Adrian C.
Published: July 2010

Abstract: Carbon fiber microelectrodes either were implanted directly into striatal tissue or were mounted into the outlet of microdialysis probes that were implanted into striatal tissue. This allowed voltammetry and microdialysis to be used under identical in vivo experimental conditions to monitor extracellular dopamine levels during electrical stimulation of the medial forebrain bundle both before and after uptake inhibition with nomifensine. The marked differences between the results obtained with each technique cannot be explained on the basis of their inherent analytical attributes (sensitivity, temporal response, etc.). The results demonstrate that the microdialysis recovery factor for endogenous dopamine increases after uptake inhibition, an observation that stands in contradiction to the existing theory and practice of the microdialysis technique. The observations led to the development of a numerical model that rationalizes the observations reported herein and that allows in vivo voltammetry and in vivo microdialysis results to be interpreted within a single theoretical framework.
Opioid Modulation of Extracellular Signal‐Regulated Protein Kinase Activity Is Ras‐Dependent and Involves Gβγ Subunits
Authors: Belcheva Mariana M. , Vogel Zvi , Ignatova Elena , Avidor‐Reiss Tomer , Zippel Renata , Levy Rivka , Young Eric C. , Barg Jacob , Coscia Carmine J.
Published: July 2010

Abstract: Although it is well‐established that G protein‐coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal‐regulated protein kinase (ERK) in African green monkey kidney COS‐7 cells transiently cotransfected with μ‐, δ‐, or κ‐opioid receptors and ERK1‐ or ERK2‐containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ‐ ([d‐Ala2,Me‐Phe4,Gly‐ol5]enkephalin)‐, δ‐ ([d‐Pen2,d‐Pen5]enkephalin)‐, or κ‐ (U69593)‐selective opioid agonists, a dose‐dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17‐Ras or with a βγ scavenger, CD8‐β‐adrenergic receptor kinase‐C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2‐h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8‐β‐adrenergic receptor kinase‐C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor‐induced ERK activity.
Role of Acetylcholine Receptors and Dopamine Transporter in Regulation of Extracellular Dopamine in Rat Carotid Body Cultures Grown in Chronic Hypoxia or Nicotine
Authors: Jackson Adele , Nurse Colin A.
Published: July 2010

Abstract: Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen‐sensitive glomus cells grown for ∼12 days in normoxia (Nox; 20% O2), chronic hypoxia (CHox; 6% O2), or chronic nicotine (CNic; 10 µM nicotine, 20% O2), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase‐positive glomus cells present, was augmented by acute (∼15‐min) exposure to hypoxia (5% O2; ∼6× basal), high extracellular K+ (30 mM; ∼10× basal), nomifensine (1 µM; a selective DA uptake inhibitor; ∼3× basal), and nicotine (100 µM; ∼5× basal), but not methylcholine (300 µM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (∼9× control), the stimulatory effect of high K+ (3–4× basal) and acute hypoxia (∼2× basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (∼9× control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (∼2× basal), nicotine, nomifensine, and high K+ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 µM), a nicotinic AChR (nAChR) antagonist. In addition, long‐term (16‐h), but not acute (15‐min), treatment with the muscarinic antagonist atropine (1 µM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.
Endogenously Occurring β‐Carboline Induces Parkinsonism in Nonprimate Animals: A Possible Causative Protoxin in Idiopathic Parkinson's Disease
Authors: Matsubara Kazuo , Gonda Tatsuo , Sawada Hideyuki , Uezono Takashi , Kobayashi Yuta , Kawamura Takashi , Ohtaki Ko‐ichi , Kimura Kojiro , Akaike Akinori
Published: July 2010

Abstract: To examine whether simple β‐carbolines induce parkinsonian‐like symptoms in vivo via N‐methylation, the simple β‐carbolines norharman (NH), 2‐mono‐N‐methylated norharmanium cation (2‐MeNH+), and 9‐mono‐N′‐methylnorharman (9‐MeNH) were systematically administered to C57BL/6 mice for 7 days. These substances induced bradykinesia with reduction of locomotion activity. NH or 2‐MeNH+ decreased dopamine (DA) contents to 50–70% of values in controls in the striatum and midbrain. 9‐MeNH potently decreased not only DA but also serotonin content in various regions. Immunohistochemical examination revealed that the numbers of tyrosine hydroxylase (TH)‐positive cells in the substantia nigra pars compacta of NH‐ and 9‐MeNH‐treated mice were diminished to 76 and 66% of values in control mice, respectively. The formation of a toxic metabolite, 2,9‐di‐N,N′‐methylated norharmanium cation (2,9‐Me2NH+), was 14 and eight times higher in the brain of mice receiving 9‐MeNH than that in NH‐ and 2‐MeNH+‐treated mice, respectively. In cultured mesencephalic cells from rat embryo, 2,9‐Me2NH+ selectively killed TH‐positive neurons only at a lower dose but was toxic to all neurons at higher doses. Thus, the excess formation of 2,9‐Me2NH+ would induce nonspecific neurotoxicity. These results indicated that 9‐indole nitrogen methylation should be the limiting step in the development of the toxicity. NH, a selective dopaminergic toxin precursor, is sequentially methylated to form 2,9‐Me2NH+, which could be an underlying factor in idiopathic Parkinson's disease.
Nicotine Exposure During a Critical Period of Development Leads to Persistent Changes in Nicotinic Acetylcholine Receptors of Adult Rat Brain
Authors: Miao H. , Liu C. , Bishop K. , Gong Z.‐H. , Nordberg A. , Zhang X.
Published: July 2010

Abstract: Effects of neonatal nicotine exposure on the development of nicotinic acetylcholine receptor (nAChR) α2, α3, α4, α7, and β2 subunit mRNAs and the number of nAChR isoforms in rat brain were studied. The mRNA levels for nAChR subunits were measured by ribonuclease protection assay, and the number of nAChR isoforms was measured with (−)‐[3H]nicotine, [3H]epibatidine, and α‐[3H]bungarotoxin ([3H]α‐Bgt). Pups were divided into two groups: One group received (−)‐nicotine treatment (0.1 mg/kg s.c. free base twice per day) during postnatal day (P) 1 to P21 and the other during P8 to P16. The period from P8 to P16 was chosen due to persistent changes that occur in brain nAChRs and in the behavior of adult mice that received (−)‐nicotine treatment during P10 to P16. (−)‐Nicotine exposure from P1 to P21 significantly up‐regulated the number of [3H]‐epibatidine and high‐affinity (−)‐[3H]nicotine binding sites in most of the brain regions studied but did not influence the number of [3H]α‐Bgt binding sites. This effect was a transient one: The up‐regulated binding sites returned to control level 1 week after withdrawal from nicotine. (−)‐Nicotine exposure during P8 to P16 resulted in a significant and long‐lasting increase in the number of nAChR isoforms labeled by (−)‐[3H]nicotine, but not by [3H]epibatidine, in the cortex, hippocampus, and striatum of adult rat. This treatment converted the low‐affinity binding sites of (−)‐nicotine into a high‐affinity state revealed by the competition studies of (−)‐[3H]nicotine/(−)‐nicotine. No changes in the mRNA levels of the subunits studied were observed following nicotine treatment during these two periods. These results suggest that the second postnatal week is a critical period during which nicotine treatment can induce permanent effects on the nAChRs in rat brain. The underlying mechanisms involved in the up‐regulation of the number of nAChRs observed in this study are posttranscriptional.
Partial Inhibition of Brain Succinate Dehydrogenase by 3‐Nitropropionic Acid Is Sufficient to Initiate Striatal Degeneration in Rat
Authors: Brouillet Emmanuel , Guyot Marie‐Caroline , Mittoux Vincent , Altairac Séverine , Condé Françoise , Palfi Stéphane , Hantraye Philippe
Published: July 2010

Abstract: Chronic inhibition of succinate dehydrogenase (SDH) by systemic injection of the selective inhibitor 3‐nitropropionic acid (3NP) has been used as an animal model for Huntington's disease (HD). However, the mechanisms by which 3NP produces lesions in the striatum are not fully characterized. A quantitative histochemical method was developed to study the level of regional SDH inhibition resulting from intraperitoneal injection of 3NP or chronic intoxication using osmotic pumps. The results showed that (a) 3NP was an irreversible SDH inhibitor in vivo, (b) the level of SDH inhibition in the striatum (the brain region most vulnerable to 3NP) was similar to that observed in other brain regions not affected by the toxin, such as the cerebral cortex, and (c) the neurotoxic threshold of SDH inhibition in the brain was 50–60% of control levels. The present study demonstrates that the selective degeneration in the striatum observed after chronic 3NP administration cannot be ascribed to a preferential inhibition of SDH in this particular brain region. This work also suggests that the partial decrease in the activity of the respiratory chain complex II–III reported in HD patients may be sufficient to induce the selective striatal degeneration observed in this disorder.
Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione
Authors: Dringen Ralf , Verleysdonk Stephan , Hamprecht Bernd , Willker Wieland , Leibfritz Dieter , Brand Annette
Published: July 2010

Abstract: The metabolism of [2‐13C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13C NMR spectroscopy. After a 24‐h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2‐13C]glycine, cell extracts and incubation media were analyzed for 13C‐labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2‐13C]glycine, the amount of de novo synthesized [2‐13C]creatine was two‐fold increased, and in addition, 13C‐labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2‐13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13C‐labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2‐13C]serine, [3‐13C]serine, and [2,3‐13C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.
Regulation of n‐3 and n‐6 Fatty Acid Metabolism in Retinal and Cerebral Microvascular Endothelial Cells by High Glucose
Authors: Delton‐Vandenbroucke Isabelle , Grammas Paula , Anderson Robert E.
Published: July 2010

Abstract: The present study was undertaken to determine whether polyunsaturated fatty acid metabolism is affected by high glucose levels in cerebral and retinal microvascular endothelial cells. The metabolism of [3‐14C]22:5n‐3 and [1‐14C]18:2n‐6 was studied in cells previously cultured for 5 days in normal (5 mM) or high (30 mM) glucose medium. After incubation of retinal endothelial cells with [3‐14C]22:5n‐3 in the high glucose condition, the formation of labeled 24:6n‐3 and 22:6n‐3 was increased, and that of labeled 24:5n‐3 was decreased, compared with the normal glucose condition. The changes were found for fatty acids esterified in cellular lipids and those released into the medium. After incubation with [1‐14C]18:2n‐6, levels of all elongation/desaturation products were increased at the expense of the precursor in retinal endothelial cells cultured in high glucose medium. The changes were primarily found for esterified fatty acids, with the release of n‐6 fatty acids being minor in both glucose concentrations. By contrast, high glucose levels did not affect the metabolism of [3‐14C]22:5n‐3 and [1‐14C]18:2n‐6 in cerebral endothelial cells. The changes in metabolic activity of retinal endothelial cells were not reflected in the fatty acid composition. The present data suggest that high glucose can increase the desaturation process in retinal but not cerebral endothelial cells. This may produce some lipid abnormalities in retinal microvasculature and contribute to altered vascular function observed in diabetic retinopathy.
Inhibition of Glycosphingolipid Biosynthesis Does Not Impair Growth or Morphogenesis of the Postimplantation Mouse Embryo
Authors: Brigande John V. , Platt Frances M. , Seyfried Thomas N.
Published: July 2010

Abstract: Whole embryo culture (WEC) of organogenesis‐stage mouse embryos was adapted for glycosphingolipid (GSL) metabolic studies to evaluate the hypothesis that de novo GSL biosynthesis is a prerequisite for growth and morphogenesis of the early postimplantation embryo. WEC supports the growth and development of postimplantation mouse embryos to stages that are indistinguishable from those achieved in vivo. N‐Butyldeoxygalactonojirimycin (NB‐DGJ) is an N‐alkylated imino sugar that specifically inhibits biosynthesis of all glucosylceramide‐based GSLs. NB‐DGJ inhibited glucosylceramide and lactosylceramide biosynthesis nearly completely and inhibited ganglioside biosynthesis ∼90% in both the embryo and visceral yolk sac. NB‐DGJ also significantly reduced total ganglioside content in both the embryo and visceral yolk sac as estimated by the cholera toxin immunooverlay technique. A shift in expression from the structurally simple to the structurally complex gangliosides was also observed in NB‐DGJ‐treated embryos and yolk sacs. Despite causing major changes in GSL biosynthesis and composition, NB‐DGJ had no effect on embryo viability, growth, or morphology. The findings suggest that de novo GSL biosynthesis may not be a prerequisite for the growth and morphogenesis of the organogenesis‐stage mouse embryo.
Identification of P2X7 (P2Z) Receptor in N18TG‐2 Cells and NG108‐15 Cells
Authors: Kaiho Hiromi , Matsuoka Isao , Kimura Junko , Nakanishi Hironori
Published: July 2010

Abstract: ATP activates a nonselective cation current by stimulating the P2Z receptor in NG108‐15 cells—a hybrid cell line of the mouse neuroblastoma N18TG‐2 cells and the rat glioma C6Bu‐1 cells. Recently, the P2X7 receptor was cloned from the rat brain and was found to have electrophysiological properties similar to those of the P2Z receptor. We examined the expression of P2X7 receptor mRNA in NG108‐15 cells as well as in their parent cell lines, N18TG‐2 and C6Bu‐1 cells, by reverse transcription‐polymerase chain reaction (RT‐PCR). The cDNA templates from these cell lines were amplified with primers specific to the P2X7 receptor sequence. Positive signals were detected in the RT‐PCR products from NG108‐15 and N18TG‐2 cells but not from C6Bu‐1 cells. We next examined the effect of ATP on the membrane current in N18TG‐2 cells and C6Bu‐1 cells by whole‐cell voltage clamp. In N18TG‐2 cells, ATP induced a sustained current with a reversal potential of 9.3 ± 1.2 mV (n = 22) in a concentration‐dependent manner with an EC50 of 1.76 ± 0.18 mM (n = 36). In contrast, ATP (1 mM) did not induce any current in C6Bu‐1 cells. The ATP‐induced current in N18TG‐2 cells resembled that in NG108‐15 cells in the following points: (a) The currents did not desensitize significantly. (b) EC50 values of ATP are of millimolar order. (c) Benzoylbenzoyl‐ATP was a more potent agonist than ATP. (d) The current was larger in methanesulfonate than in Cl− external solution. (e) The current was larger at lower external Mg2+ concentrations. These results suggest that the hybrid NG108‐15 cells possess a P2X7 receptor like the P2Z receptor and that the ability of expressing this channel originates from N18TG‐2 cells but not from C6Bu‐1 cells.
Modulation of Human Glutamate Transporter Activity by Phorbol Ester
Authors: Ganel Raquelli , Crosson Craig E.
Published: July 2010

Abstract: Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high‐affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na+‐dependent mammalian glutamate transporters exist. Our study demonstrates that Y‐79 human retinoblastoma cells express a single Na+‐dependent glutamate uptake system with a Km of 1.7 ± 0.42 µM that is inhibited by dihydrokainate and dl‐threo‐β‐hydroxyaspartate (IC50 = 0.29 ± 0.17 µM and 2.0 ± 0.43 µM, respectively). The protein kinase C activator phorbol 12‐myristate 13‐acetate caused a concentration‐dependent inhibition of glutamate uptake (IC50 = 0.56 ± 0.05 nM), but did not affect Na+‐dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the Vmax. 4α‐Phorbol 12,13‐didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12‐myristate 13‐acetate‐induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y‐79 cell line indicates that it belongs to the dihydrokainate‐sensitive EAAT2/GLT‐1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.
α‐Ketoisocaproate Alters the Production of Both Lactate and Aspartate from [U‐13C]Glutamate in Astrocytes: A 13C NMR Study
Authors: McKenna Mary C. , Sonnewald Ursula , Huang Xueli , Stevenson Joseph , Johnsen Svein F. , Sande Leif M. , Zielke H. Ronald
Published: July 2010

Abstract: The present study determined the metabolic fate of [U‐13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mMα‐ketoisocaproate (α‐KIC). When astrocytes were incubated with 0.2 mM [U‐13C]glutamate, 64.1% of the 13C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3‐13C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [13C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [13C]glutamate into aspartate in astrocytes incubated in the presence of α‐KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [13C]glutamate into the 1,2,3‐13C3‐isotopomer of lactate in cells incubated in the presence of α‐KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of α‐KIC. Altogether more of the [13C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of α‐KIC than in control cells. Overall, the results demonstrate that the presence of α‐KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate‐aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.
Differential Involvement of Metabotropic and p75 Neurotrophin Receptors in Effects of Nerve Growth Factor and Neurotrophin‐3 on Cultured Purkinje Cell Survival
Authors: Mount Howard T. J. , Elkabes Stella , Dreyfus Cheryl F. , Black Ira B.
Published: July 2010

Abstract: We have examined the role of the p75 neurotrophin receptor in survival‐promoting effects of nerve growth factor (NGF) and neurotrophin‐3 (NT‐3) on cultured Purkinje cells. Previously, we showed that NGF promotes Purkinje cell survival in conjunction with (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACPD), an agonist of metabotropic excitatory amino acid receptors, whereas NT‐3 by itself increases cell number. We now present evidence that p75 plays different roles in Purkinje cell responses to the two neurotrophins. A metabotropic receptor of the mGluR1 subtype may interact with p75 function, so as to regulate Purkinje cell responsiveness to neurotrophins. When cerebellar cultures were grown for 6 days in the presence of ACPD and a mutant form of NGF that does not bind to p75, no increase in Purkinje cell number was observed. Moreover, the survival‐promoting effect of wild‐type NGF and ACPD could be inhibited by a neutralizing antiserum to p75 or by a pyrazoloquinazolinone inhibitor of neurotrophin binding to p75. In contrast, the response to NT‐3 was potentiated by anti‐p75 treatment and by the quinazolinone. These data indicate the mediation of p75 in the trophic response to NGF‐ACPD and a negative modulatory role of p75 in the action of NT‐3. To probe the role of ACPD in the p75‐dependent response to NGF, metabotropic receptor subtype‐specific ligands were tested. The pattern of agonist specificity implicated the mGluR1 subtype, a receptor that is expressed at high levels by Purkinje cells and linked to activation of protein kinase C (PKC). Down‐regulation or blockade of PKC abolished the response to NGF‐ACPD. Consistent with the opposite roles of p75 in effects of the two neurotrophins, blockade of mGluR1 or PKC potentiated the survival response elicited by NT‐3. In sum, our data suggest that afferent excitatory transmitters activate specific metabotropic receptors to elicit a p75‐mediated action of NGF. NT‐3 acts on Purkinje cells by a different mechanism that is not absolutely p75‐dependent and that is reduced by neurotrophin access to p75 and metabotropic receptor activity.
Release and Uptake Rates of 5‐Hydroxytryptamine in the Dorsal Raphe and Substantia Nigra Reticulata of the Rat Brain
Authors: Bunin Melissa A. , Prioleau Cassandra , Mailman R. B. , Wightman R. Mark
Published: July 2010

Abstract: Fast scan cyclic voltammetry with carbon fiber electrodes has been used to investigate the dynamics of the neurotransmitter 5‐hydroxytryptamine (5‐HT) in the extracellular fluid of two brain regions: the dorsal raphe and the substantia nigra reticulata. The method used previously was shown to be optimized to allow the time course of 5‐HT concentration changes to be measured rapidly. Measurements were made in slices prepared from the brains of rats with the carbon fiber electrode inserted into the tissue and a bipolar stimulating electrode placed on the slice surface. Identification of 5‐HT as the detected substance in both regions was based on voltammetric, anatomical, physiological, and pharmacological evidence. Autoradiography using [3H]paroxetine revealed highest 5‐HT transporter binding densities in the regions in which voltammetric measurements were made. Evaluation of the pharmacological actions of tetrodotoxin and tetrabenazine, as well as the effects of calcium removal, suggested that 5‐HT storage was vesicular and that the release process was exocytotic. The effects of fluoxetine (0.5 µM) were typical of a competitive uptake inhibitor, changing Km with little effect on Vmax. Release of 5‐HT was found to be maximal with wide (2‐ms) stimulus pulses in both regions, as expected for release from small unmyelinated processes, and to increase linearly with the number of pulses when high frequencies (100 Hz) were used. At lower frequencies, the concentration observed was a function of both release and uptake. Kinetic simulations of the data revealed that the major difference in 5‐HT neurotransmission between the two regions was that release and uptake rates are twice as large in the dorsal raphe ([5‐HT] per pulse = 100 ± 20 nM, Vmax = 1,300 ± 20 nM/s for dorsal raphe; [5‐HT] per pulse = 55 ± 7 nM, Vmax = 570 ± 70 nM/s for substantia nigra reticulata). When normalized to tissue content, uptake rates in both regions were identical and similar to rates previously reported for dopamine in dopamine terminal regions. Nonetheless, compared with dopaminergic transmission in terminal regions such as the striatum, the absolute clearance rates in the substantia nigra reticulata and dorsal raphe were lower, resulting in a longer lifetime of 5‐HT in the extracellular fluid and allowing long‐range interactions.
Arecoline Desensitizes Carbachol‐Stimulated Phosphatidylinositol Breakdown in Rat Brain Cortices
Authors: Lee Horng‐Mo , Tsai Kuen‐Jer , Lin Chien‐Huang , Huang Chung‐Lin , Tung Che‐Se
Published: July 2010

Abstract: To understand the effects of arecoline administration on the muscarinic cholinergic signaling pathway, rats were injected with arecoline, 10 mg/kg i.p., and the carbachol‐stimulated phosphoinositide breakdown in rat brain cortical slices was examined. In vivo administration of arecoline resulted in inhibition of carbachol‐stimulated phosphoinositide turnover in rat brain cortical slices. Arecoline was a partial agonist with peak effects of 30% of the maximum as obtained with carbachol. Coaddition of arecoline inhibited the carbachol‐stimulated phosphoinositide breakdown. Pretreatment of rat brain cortical slices with arecoline in vitro resulted in a dose‐dependent inhibition of carbachol‐stimulated [3H]inositol monophosphate accumulation. The inhibition occurred rapidly, with half‐maximal inhibition occurring at 15 min and maximal inhibition achieved within 60 min. The inhibition of phosphoinositide breakdown was recovered 1 h after arecoline was removed. When synaptoneurosomes were used for the ligand binding studies, arecoline pretreatment was found to have decreased the maximal ligand binding (Bmax) without inducing any marked change in binding affinity (KD). The influence could be recovered by incubating the synaptoneurosomes in the absence of arecoline for 2 h. Taken together, these data suggest that the underlying mechanism by which phosphoinositide turnover is inhibited is arecoline‐induced receptor sequestration.
Glucocorticoids Provoke a Shift from α2‐ to α1‐Adrenoreceptor Activities in Cultured Hypothalamic Slices Leading to Opposite Noradrenaline Effect on Corticotropin‐Releasing Hormone Release
Authors: Feuvrier Emmanuelle , Aubert Magali , Mausset Anne‐Laure , Alonso Gérard , Gaillet Sylvie , Malaval Francis , Szafarczyk Alain
Published: July 2010

Abstract: We have shown previously that noradrenaline (NA) stimulated or inhibited the release of corticotropin‐releasing hormone (CRH) according to the availability of adrenal steroids. The aim of the present work was to examine whether the changes in the NA modulation of CRH release from hypothalamic neurons result from a steroid‐induced plasticity of the adrenergic transduction pathways. From anterior hypothalamic slices cultured in standard medium (i.e., containing adrenal steroids at a final dilution of 61 ± 9 ng/ml), (a) the stimulatory effect of NA on CRH release was reversed in a dose‐dependent manner by increasing concentrations of the α1‐adrenoreceptor antagonist prazosin, (b) activation of protein kinase C by acute treatment with phorbol 12‐myristate 13‐acetate (0.5 µM, 1 h) mimicked NA stimulation of CRH secretion, and (c) the activation of L‐type Ca2+ channels by Bay K 8644 also produce an increased CRH secretion. In contrast, the inhibitory effect of NA on CRH secretion from slices cultured in steroid‐free medium was markedly reversed by the α2‐adrenoreceptor antagonist yohimbine, by pretreatment with pertussin toxin, or by the addition of 4‐aminopyridine, a K+‐channel blocker. Acute treatment with phorbol 12‐myristate 13‐acetate did not change the inhibitory NA effect. Moreover, all these effects were reversed by daily corticosterone supplementation, for as long as they were tested. These results are consistent with a steroid‐dependent change in the nature of adrenergic receptors and its associated transduction pathways involved in the regulation of CRH secretion in the hypothalamus.
Diamine Oxidase Induces Neurite Outgrowth in Chick Dorsal Root Ganglia by a Nonenzymatic Mechanism
Authors: Munis James R. , Steiner Joseph T. , Ruat Martial , Snyder Solomon H.
Published: July 2010

Abstract: The enzyme diamine oxidase (DAO) catalyzes the oxidative deamination of histamine, diamines, and polyamines. DAO has been localized to several tissues, including thymus, kidney, intestine, seminal vesicles, placenta, and pregnancy plasma. DAO is not constitutively expressed in the mammalian brain, but it becomes detectable following focal injury. Although the physiologic role of DAO remains unknown, the observation that it is present at the interface between rapidly dividing and quiescent cells in several tissues suggests that it might be involved in regulating cell division or differentiation at tissue boundaries. In addition, the observation that DAO is expressed in the brain following injury suggests that the protein might play a role in the CNS response to focal neuronal damage. To test that hypothesis, we assessed the ability of purified DAO to alter the pattern of neuronal differentiation and nerve growth in vitro. In chick dorsal root ganglion explant cultures, purified porcine DAO induced neurite outgrowth in the low nanomolar range. Addition of aminoguanidine, which inhibits DAO enzyme activity, did not inhibit the protein's neurotrophic activity. These findings suggest that DAO can function as a neurotrophic ligand independent of its enzymatic activity.
Splicing of a Regulated Exon Reveals Additional Complexity in the Axonal Microtubule‐Associated Protein Tau
Authors: Wei Mei‐Ling , Andreadis Athena
Published: July 2010

Abstract: Tau is a microtubule‐associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression pattern and splicing regulation had not been previously analyzed in the human. The expression profile of exon 6 is completely different from that of the better‐analyzed exons 2, 3, 4A, and 10, implying the utilization of distinct regulatory factors. The default splicing behavior of the exon had demonstrated the existence of what were initially considered cryptic splice sites. However, analysis of the expression pattern of exon 6 suggests that these splice sites are utilized in certain human tissues and, if translated, would result in a radically altered tau protein. Lastly, expression of exon 6 minigene constructs in cells indicates that its flanking exons are involved in its inclusion and in the modulation of the ratio of its variants.
Collaborative and Reciprocal Effects of Ciliary Neurotrophic Factor and Nerve Growth Factor on the Neuronal Phenotype of Human Neuroblastoma Cells
Authors: Zinman L. H. , Lawrance G. , Wang W. , Verge V. M. K. , Dow K. E. , Maurice D. H. , Richardson P. M. , Riopelle R. J.
Published: July 2010

Abstract: We have probed the molecular basis of functional effects of ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) on aspects of the neuronal differentiation of LA‐N‐2 neuroblastoma cells. The influence of CNTF on the cholinergic phenotype can be accounted for by transcriptional/translational effects without implicating posttranslational mechanisms. Although both NGF receptors are expressed constitutively by LA‐N‐2 cells, CNTF has a marked stimulatory effect on trkA mRNA and protein. The NGF receptors are functional in serum‐free conditions where they mitigate CNTF effects on cell adhesion but do not support process extension. Following priming by CNTF, NGF and CNTF have synergistic influences on process formation but not on choline acetyltransferase‐specific activity.
Continuous Activation of Pituitary Adenylate Cyclase‐Activating Polypeptide Receptors Elicits Antipodal Effects on Cyclic AMP and Inositol Phospholipid Signaling Pathways in CATH.a Cells: Role of Protein Synthesis and Protein Kinases
Authors: Muller André , Lutz‐Bucher Bernadette , Kienlen‐Campard Pascal , Koch Bernard , Loeffler Jean‐Philippe
Published: July 2010

Abstract: Continuous exposure of cells to agonists develops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron‐like cell line), which express pituitary adenylate cyclase‐activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C‐β pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down‐regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP‐stimulated cyclic AMP accumulation was attenuated in a time‐ and dose‐dependent fashion (t1/2 = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also unchanged in the presence of 3‐isobutyl‐1‐methylxanthine, indicating that neither Gi‐like proteins nor cyclic nucleotide phosphodiesterases play a critical role in these processes. Blockade of protein synthesis with cycloheximide, as well as inhibition by H89 of cyclic AMP‐dependent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phosphate formation. Transcription of the chimeric GAL4‐CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was potentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross‐talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP‐dependent protein kinase activation. At the same time, they show that desensitization of cyclic AMP signaling not only fails to hamper, but actually amplifies PACAP‐stimulated CREB‐regulated transcription.
Down‐Regulation of the Noradrenaline Transporter by Interferon‐α in Cultured Bovine Adrenal Medullary Cells
Authors: Toyohira Yumiko , Yanagihara Nobuyuki , Minami Kouichiro , Ueno Susumu , Uezono Yasuhito , Tachikawa Eiichi , Kondo Yukiko , Kashimoto Takeshi , Izumi Futoshi
Published: July 2010

Abstract: The aim of this study was to investigate the effect of long‐term treatment with interferon (IFN)‐α on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN‐α caused a decrease in uptake of [3H]noradrenaline by the cells in time (4–48 h)‐ and concentration (300–1,000 U/ml)‐dependent manners. IFN‐β also inhibited [3H]noradrenaline uptake to a lesser extent than did IFN‐α, whereas IFN‐γ had little effect. An anti‐IFN‐α antibody reduced the effect of IFN‐α on [3H]noradrenaline uptake. Saturation analysis of [3H]noradrenaline uptake showed that the inhibitory effect of IFN‐α was due to a reduction in the maximal uptake velocity (Vmax) values without altering apparent Michaelis constant (Km) values. Incubation of cells with IFN‐α caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN‐α on [3H]noradrenaline uptake was diminished in protein kinase C‐down‐regulated cells. Incubation of cells with IFN‐α for 48 h significantly reduced the specific binding of [3H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [3H]desipramine binding revealed that IFN‐α decreased the maximal binding (Bmax) values without any change in the dissociation constant (KD) values. These findings suggest that IFN‐α suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.
Repeated Cocaine Administration Alters Extracellular Glutamate in the Ventral Tegmental Area
Authors: Kalivas Peter W. , Duffy Patricia
Published: July 2010

Abstract: The present study determined if repeated cocaine injections alter the effect of cocaine on extracellular glutamate in the ventral tegmental area (VTA). All rats were treated with daily cocaine (15 mg/kg i.p. × 2 days, 30 mg/kg i.p. × 5 days) or saline for 7 days. At 21 days after discontinuing the daily injections, a dialysis probe was placed into the VTA and the extracellular levels of glutamate were estimated. A systemic injection of cocaine (15 mg/kg i.p.) elevated extracellular glutamate in the VTA of rats pretreated with daily cocaine but not in the daily saline‐pretreated subjects. No significant change in glutamate was produced by a saline injection in either pretreatment group. In a group of rats pretreated with daily cocaine, the D1 antagonist SCH‐23390 (30 µM) was infused through the dialysis probe prior to the acute injections of saline and cocaine. SCH‐23390 prevented the increase in extracellular glutamate associated with the acute administration of cocaine. Behavioral data were collected simultaneously with the measures of extracellular glutamate. The behavioral stimulant effect of cocaine was greater in cocaine‐pretreated than saline‐pretreated subjects, and the behavioral augmentation in cocaine‐pretreated rats was partly blocked by SCH‐23390. These data support the hypotheses that repeated cocaine administration produces an increase in the capacity of D1 receptor stimulation to release glutamate in the VTA and that this mechanism partly mediates behavioral sensitization produced in rats treated with daily cocaine injections.
Increase of Extracellular Glutamate and Expression of Fos‐Like Immunoreactivity in the Ventral Tegmental Area in Response to Electrical Stimulation of the Prefrontal Cortex
Authors: Rossetti Zvani L. , Marcangione Caterina , Wise Roy A.
Published: July 2010

Abstract: Electrical stimulation of the medial prefrontal cortex caused glutamate release in the ventral tegmental area (VTA) of freely moving animals. Cathodal stimulation was given through monopolar electrodes in 0.1‐ms pulses at an intensity of 300 µA and frequencies of 4–120 Hz. Glutamate was measured in 10‐min perfusate samples by HPLC coupled with fluorescence detection following precolumn derivatization with o‐phthaldialdehyde/β‐mercaptoethanol. The stimulation‐induced glutamate release was frequency dependent and was blocked by the infusion of the sodium channel blocker tetrodotoxin (10 µM) through the dialysis probe. The stimulation also induced bilateral Fos‐like immunoreactivity in ventral tegmental neurons, with a significantly greater number of Fos‐positive cells on the stimulated side. These findings add to a growing body of evidence suggesting that the medial prefrontal cortex regulates dopamine release in the nucleus accumbens via its projection to dopamine cell bodies in the VTA.
Down‐Regulation of the Human Norepinephrine Transporter in Intact 293‐hNET Cells Exposed to Desipramine
Authors: Zhu Meng‐Yang , Blakely Randy D. , Apparsundaram Subramanian , Ordway Gregory A.
Published: July 2010

Abstract: The effects of continuous exposure of cultured cells expressing the human norepinephrine transporter (hNET) to the hNET inhibitor desipramine on hNET expression and function were studied. Exposure of HEK‐293 cells transfected stably with the hNET cDNA (293‐hNET cells) to desipramine for 3 days reduced the specific binding of [3H]nisoxetine in membrane homogenates in a concentration‐dependent manner. The magnitude of the reductions in [3H]nisoxetine binding to hNET was dependent on the length of time of the exposure to desipramine, reaching 77% after a 21‐day exposure. The reduction of [3H]nisoxetine binding returned to control levels within 72 h after a 3‐day exposure to desipramine. Reductions in [3H]nisoxetine binding to hNET were accompanied by time‐dependent and exposure concentration‐dependent reductions in hNET protein levels as determined by western blotting. Similar to binding, hNET protein levels returned to control levels 72 h after cessation of desipramine exposure. Northern blotting indicated that exposure of 293‐hNET cells to desipramine did not significantly alter hNET mRNA levels. Uptake of [3H]norepinephrine by 293‐hNET cells was markedly reduced after a 3‐day exposure to desipramine. However, desipramine exposure had no effect on uptake of [3H]glutamate or [3H]‐alanine. The present findings imply that down‐regulation of the hNET in 293‐hNET cells induced by desipramine results from a selective reduction in hNET protein levels, presumably a consequence of either a reduction in the translation of hNET mRNA or from an enhanced degradation of hNET protein.
Distinct Changes in Peptide YY Binding to, and mRNA Levels of, Y1 and Y2 Receptors in the Rat Hippocampus Associated with Kindling Epileptogenesis
Authors: Gobbi M. , Gariboldi M. , Piwko C. , Hoyer D. , Sperk G. , Vezzani A.
Published: July 2010

Abstract: Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of 125I‐peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% (p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham‐stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36–87% (p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50–64% (p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% (p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y‐mediated neurotransmission.
Tetanus Toxin Enhances Protein Kinase C Activity Translocation and Increases Polyphosphoinositide Hydrolysis in Rat Cerebral Cortex Preparations
Authors: Gil Carles , Ruiz‐Meana Marisol , Álava María , Yavin Ephraim , Aguilera José
Published: July 2010

Abstract: Tetanus toxin (TeTx) has been recently demonstrated to be a Zn2+‐dependent endopeptidase that cleaves synaptobrevin, a protein in part responsible for neurotransmitter release. Nevertheless, certain aspects of TeTx action, for example, the causal relationship between TeTx and protein kinase C (PKC; EC 2.7.1.37) activity cannot be explained by this cleavage alone. In the present study, primary neurons from fetal rat brain, synaptosomes, and whole slices have been used to examine this issue. Low doses of TeTx (≤ 10−8M) caused PKC activity translocation in a manner similar to that produced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). TPA (≤ 10−7M) caused sustained PKC activity translocation, whereas TeTx produced translocation followed by relocation, depending on the dose and time of exposure. Immunoidentification with a monoclonal antibody recognizing both α and β isoforms revealed that TeTx induced moderate losses of PKC in the cytosolic fraction, without a comparable increase in the particulate fraction. Although moderate losses of activity were also noticed in the cytosolic fraction, the inconsistency with respect to activity translocation may be explained by translocation of additional PKC isoforms that are not identified by the antibody. Comparable levels of water‐soluble inositol phosphate‐labeled intermediates were obtained after treatment of cerebral cells and/or cortical brain slices with TeTx. Significant increases of 19 and 114% in the water‐soluble myo‐[2‐3H]inositol‐labeled inositol phosphate metabolites were found in cerebral cell culture and brain slices, respectively, after treatment with 10−8M TeTx. TeTx (10−8M) increased to the same degree the water‐soluble inositol phosphate levels as did serotonin (10−5M) or carbachol (10−6M). It is suggested that part of the signaling cascade of TeTx consists of a component involving inositol phospholipid hydrolysis, which is associated with PKC activity translocation.