bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution and Developmental Regulation of Metabotropic Glutamate Receptor 7a in Rat Brain
Authors: Bradley Stefania Risso , Rees Howard D. , Yi Hong , Levey Allan I. , Conn P. Jeffrey
Published: July 2010

Abstract: To determine the regional and cellular distribution of the metabotropic glutamate receptor mGluR7a, we used rabbit anti‐peptide polyclonal‐targeted antibodies against the C‐terminal domain of mGluR7a. Here we report that immunocytochemistry at the light‐microscopic level revealed that mGluR7a is widely distributed throughout the adult rat brain, with a high level of expression in sensory areas, such as piriform cortex, superior colliculus, and dorsal cochlear nucleus. In most brain structures, mGluR7a immunoreactivity is characterized by staining of puncta and fibers. However, in some regions, including the locus ceruleus, cerebellum, and thalamic nuclei, both cell bodies and fibers are immunopositive. The changes in levels of mGluR7a during development were investigated with immunoblotting and immunocytochemical analysis. Immunoblot analysis revealed that the levels of mGluR7a are differentially regulated across brain regions during postnatal development. In cortical regions (hippocampus, neocortex, and olfactory cortex), mGluR7a levels were highest at postnatal day 7 (P7) and P14, then declined in older rats. In contrast, mGluR7a levels were highest at P7 in pons/medulla and cerebellum and decreased markedly between P7 and P14. In these regions, mGluR7a immunoreactivity was at similar low levels at P14 and P21 and in adults. Immunocytochemical analysis revealed that staining for mGluR7a was exceptionally high in fiber tracts in P7 animals relative to adults. Furthermore, the pattern of mGluR7a immunoreactivity in certain brain structures, including cerebellum, piriform cortex, and hippocampus, was significantly different in P7 and adult animals. In summary, these data suggest that mGluR7a is widely distributed throughout the rat brain and that this receptor undergoes a dynamic, regionally specific regulation during postnatal development.
Voltammetric Studies on Mechanisms of Dopamine Efflux in the Presence of Substrates and Cocaine from Cells Expressing Human Norepinephrine Transporter
Authors: Chen Nianhang , Trowbridge C. G. , Justice J. B.
Published: July 2010

Abstract: The effects of substrates m‐tyramine and β‐phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration‐dependent manner. Their EC50 values for inducing DA efflux were similar to their IC50 values for inhibiting DA uptake. The substrate‐induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na+] with Na+,K+‐ATPase inhibition, but was insensitive to membrane potential‐altering agents valinomycin, veratridine, and high [K+]. The initial rate of m‐tyramine‐induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis‐Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na+], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first‐order rate constant for m‐tyramine‐induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET‐dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine “uncovers” NET‐independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.
Induction of Long‐Term Potentiation In Vivo Regulates Alternate Splicing to Alter Syntaxin 3 Isoform Expression in Rat Dentate Gyrus
Authors: Rodger Jennifer , Davis Sabrina , Laroche Serge , Mallet Jacques , Hicks Andrew
Published: July 2010

Abstract: The regulation and specificity of the interactions between the proteins involved in neurotransmitter release are obvious targets for the cellular control of synaptic plasticity. Previous research has identified one of these proteins, syntaxin 1B, as a potential target for mediating the propagation of synaptic plasticity through neural networks. The expression of syntaxin 1B is modified in the hippocampus after the induction of long‐term potentiation (LTP) and during learning. Here, we describe the identification of two other members of the syntaxin family from rat brain, syntaxins 3A and 3B, and show that they are generated from the same gene by alternate splicing. In situ hybridization and immunohistochemical staining confirm the expression of syntaxins 3A and 3B in the adult rat brain. The transcripts and proteins show a lower abundance but a similar pattern of expression as syntaxins 1A and 1B. By using quantitative competitive PCR, we show that the mRNAs that encode syntaxins 1B and 3A are increased in dentate granule cells 6 h after the induction of LTP in vivo, whereas syntaxin 3B mRNA is decreased as rapidly as 30 min, and lasts for at least 6 h, after the induction of LTP. These findings identify coordinated changes in the expression of several syntaxin isoforms with different substrate specificities and suggest that regulation of the splicing machinery by LTP induction is one of the diverse strategies used during the long‐term modification of the synapse in the vertebrate nervous system.
Nitric Oxide Regulates Cyclic GMP‐Dependent Protein Kinase Phosphorylation in Rat Brain
Authors: El‐Husseini Alaa El‐Din , Bladen Christopher , Williams Julie A. , Reiner Peter B. , Vincent Steven R.
Published: July 2010

Abstract: Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP‐dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase‐dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP‐dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.
Alterations of Na,K‐ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n‐3 Fatty Acids
Authors: Gerbi A. , Maixent J.‐M. , Barbey O. , Jamme I. , Pierlovisi M. , Coste T. , Pieroni G. , Nouvelot A. , Vague P. , Raccah D.
Published: July 2010

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K‐ATPase activity. The present study in rats was designed first to measure diabetes‐induced abnormalities in Na,K‐ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil‐rich diet (rich in n‐3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n‐7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n‐6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n‐9) fatty acids. Protein expression of Na,K‐ATPase α subunits, Na,K‐ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K‐ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1‐ and α3‐isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes‐induced alterations in sciatic nerve Na,K‐ATPase activity and function.
A Culture Model of Reactive Astrocytes: Increased Nerve Growth Factor Synthesis and Reexpression of Cytokine Responsiveness
Authors: Wu Vivian W. , Nishiyama Nobuyoshi , Schwartz Joan P.
Published: July 2010

Abstract: Reactive gliosis, which occurs in response to damage to the central nervous system, has been recognized for years but is not yet understood. We describe here a tissue culture model of reactive astrocytes used to characterize their properties. Cultures are prepared 1 week following 6‐hydroxydopamine (6‐OHDA) lesion of rat substantia nigra and compared with astrocytes cultured from normal adult rats or rats injected with saline only. Astrocytes from the 6‐OHDA‐lesioned side contained elevated levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA and were intensely immunoreactive for GFAP, vimentin, and two epitopes that in vivo are found only on reactive astrocytes. The basal content of nerve growth factor (NGF) mRNA and NGF in astrocytes from 6‐OHDA‐lesioned rats was significantly higher relative to control astrocytes. Two inflammatory cytokines, interleukin‐1β and interferon‐γ, increased synthesis of NGF up to 20‐fold in the reactive cells, whereas there was no response in the normal adult astrocytes. Astrocytes from postnatal day 2 rats shared many of the properties of the reactive adult astrocytes. These cultures offer the possibility to characterize the cellular and molecular properties of reactive astrocytes and to determine the factors responsible for activation of astrocytes.
Rat Brain Type II 5′‐Iodothyronine Deiodinase Activity Is Extremely Sensitive to Stress
Authors: Baumgartner Andreas , Hiedra Luis , Pinna Graziano , Eravci Murat , Prengel Hans , Meinhold Harald
Published: July 2010

Abstract: The effects of different kinds of acute stressor on thyroid hormone concentrations and deiodinase activities were investigated in four brain regions (frontal cortex, amygdala, hypothalamus, and cerebellum) and in the pituitaries and livers of adult male rats. Five groups of rats were killed after each of the following stressors: (a) an intraperitoneal injection of saline, (b) intragastric intubation, (c) and (d) two different forms of handling, being grasped as for intraperitoneal injection and being moved from one cage to another, and (e) a 2‐h period spent in a slowly rotating drum. Two other groups were placed in the rotating drums for 10 and 19 h (sleep deprivation experiment), respectively. All stressors induced significant (in some cases up to 200%) increases in the activity of type II 5′‐iodothyronine deiodinase, which catalyzes the deiodination of the prohormone l‐thyroxine (T4) to the active metabolite 3,3′,5‐triiodo‐l‐thyronine (T3). As a consequence, the tissue concentrations of T4 fell, and those of T3 rose (sometimes by up to 300%). However, these changes were limited to selected areas of the brain that were specific for each stressor and were not seen in all brain regions investigated in any group. No clear‐cut effects of stress were seen on the activities of the type III 5‐iodothyronine deiodinase isoenzyme, which catalyzes the inactivation of T3, on liver or serum thyroid hormone concentrations or on liver of brain type I 5′‐iodothyronine deiodinase activities. In summary, our results show that even mild and very brief stress can induce marked increases in T3 concentrations specifically in brain but not in liver or blood. Thus, contrary to common opinion, thyroid hormones may play an important physiological role in stress reactions, at least in tissues that contain type II 5′‐iodothyronine deiodinase, such as brain and pituitary.
Effects of Adrenal Steroids on Basal Ganglia Neuropeptide mRNA and Tyrosine Hydroxylase Radioimmunoreactive Levels in the Adrenalectomized Rat
Authors: Lucas Louis R. , Pompei Pierluigi , Ono Jennie , McEwen Bruce S.
Published: July 2010

Abstract: To investigate the effects of type I (mineralocorticoid) and type II (glucocorticoid) receptor activation on striatal neuropeptide [preproenkephalin (PPE), preprotachykinin (PPT), and preprodynorphin (DYN)] mRNA and midbrain cholecystokinin (CCK) mRNA as well as striatal tyrosine hydroxylase radioimmunoreactivity (TH‐RIC) levels, we administered either replacement levels of corticosterone (CORT; 0.5 mg/kg/day, s.c.) or pharmacological levels of deoxycorticosterone acetate (DOCA; a mineralocorticoid steroid with ability to bind to type I and type II receptors; 5 mg/kg, s.c.) to adrenalectomized adult male rats. After 1 week of recovery from adrenalectomy surgery, animals were injected daily with sesame oil or CORT for 1, 3, or 7 days or DOCA for 3 or 7 days and killed 16 h after the last injection. Adrenalectomy resulted in a decrease in all three striatal neuropeptide mRNA levels, compared with sham‐operated rats. CORT replacement resulted in recovered PPE and PPT mRNA levels after 1 day and elevated PPE mRNA levels over those in sham‐operated controls after 3 days. In contrast, DYN mRNA levels showed recovery after 7 days of CORT replacement. Results after DOCA treatment largely paralleled those after CORT replacement. There were no significant treatment effects on indirect markers of midbrain dopaminergic activity, i.e., CCK mRNA and TH‐RIC. From these results we conclude that compared with striatal tachykinin and dynorphinergic neurons, enkephalinergic cells show greater sensitivity, whereas the dopaminergic system, including mesencephalic CCK, demonstrates an insensitivity to physiological CORT and to pharmacological DOCA treatment.
Mitochondrial Malic Enzyme: Purification from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization in Neurons of Rat Brain
Authors: Vogel R. , Jennemann G. , Seitz J. , Wiesinger H. , Hamprecht B.
Published: July 2010

Abstract: To elucidate the cellular location of mitochondrial malic enzyme in brain, immunocytochemical studies were performed. For this purpose, mitochondrial malic enzyme was purified to apparent homogeneity from bovine brain and used for the immunization of rabbits. Subjecting the antiserum to affinity purification on immobilized antigen as an absorbent yielded a purified immunoreactive antibody preparation, which was characterized by probing cytosolic and mitochondrial fractions of bovine and rat brain in western blotting. As neither crossreactivity with cytosolic malic enzyme nor immunoreactivity against other proteins could be observed, the antibody preparation was found suitable for immunocytochemistry. By using sections of perfusion‐fixed rat brain, considerable resolution was achieved at the light‐microscopic level. Distinct and specific staining of neurons was observed; in contrast, no staining of astrocytes and possibly unspecific staining within the nuclei of oligodendrocytes were obtained. From these data, it is concluded that mitochondrial malic enzyme is located in neurons; however, in astrocytes, the enzyme appears to be either lacking or present at a much lower level. A protective role against oxidative stress in neurons is proposed for mitochondrial malic enzyme.
Role of Branched‐Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism
Authors: Hutson S. M. , Berkich D. , Drown P. , Xu Baiyang , Aschner Michael , LaNoue Kathryn F.
Published: July 2010

Abstract: Because it is well known that excess branched‐chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α‐ketoglutarate to form the branched‐chain α‐keto acids (BCKAs). The brain is unique in that it expresses two separate branched‐chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte‐enriched cultures in the presence of [U‐14C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α‐ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α‐keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α‐ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.
Behavioral and Neuropathologic Changes Induced by Central Injection of Carboxyl‐Terminal Fragment of β‐Amyloid Precursor Protein in Mice
Authors: Song D.‐K. , Won M.‐H. , Jung J.‐S. , Lee J.‐C. , Kang T.‐C. , Suh H.‐W. , Huh S.‐O. , Paek S.‐H. , Kim Y.‐H. , Kim S.‐H. , Suh Y.‐H.
Published: July 2010

Abstract: Expression of the carboxyl‐terminal fragment (CT) of the β‐amyloid precursor protein (APP) in transgenic animals has been linked with neurotoxicity. However, it remains to be clarified whether the neurotoxicity is caused by β‐amyloid proteins (Aβs) derived from CT or by CT itself. To study the in vivo neurotoxicity of CT, mice were given a single intracerebroventricular injection of a recombinant 105‐amino acid CT (CT105; 68.5–685 pmol, intracerebroventricularly), and changes in behavior and in brain histology were examined. Animals given CT105 (410 or 685 pmol, intracerebroventricularly) showed a dose‐dependent impairment in the passive avoidance performance, whereas boiled CT105 had no effect. CT105 (685 pmol, intracerebroventricularly) induced reactive gliosis in neocortex and hippocampus and neurodegeneration in neocortex. These results indicate that centrally administered CT105 induces behavioral impairment and neuropathologic changes, suggesting a direct toxic effect of CT105 per se.
Cloning and Functional Characterisation of Two Novel Nicotinic Acetylcholine Receptor α Subunits from the Insect Pest Myzus persicae
Authors: Sgard F. , Fraser S. P. , Katkowska M. J. , Djamgoz M. B. A. , Dunbar S. J. , Windass J. D.
Published: July 2010

Abstract: Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor α subunits from a major insect pest, the peach‐potato aphid Myzus persicae. These two subunits, termed Mpα1 and Mpα2, are respective structural homologues of the Drosophila Dα2/Schistocerca gregariaαL1 α‐subunit pair and the Drosophila ALS α subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine‐ or nicotine‐gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick β2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist‐induced currents was observed with Mpα2 and none with Mpα1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor α subunits from different species can exhibit distinctive physiological and pharmacological characteristics.
Retinoic Acid Regulates the Developmental Expression of Dopamine D2 Receptor in Rat Striatal Primary Cultures
Authors: Valdenaire Olivier , Maus‐Moatti Marion , Vincent Jean‐Didier , Mallet Jacques , Vernier Philippe
Published: July 2010

Abstract: The time course of D2 receptor expression assessed by the levels of the corresponding binding sites and mRNA was studied in rat striatum during ontogenesis and in primary cultures of cells taken at embryonic day (E) 17 and postnatal day (P) 4. In the two experimental situations, the amount of D2 receptor mRNA and number of binding sites increased regularly from E16 to P15, indicating that expression of D2 receptors in striatal neurons occurs independently from a dopaminergic input. Incubation of striatal primary cultures with 10−5M retinoic acid significantly increased the level of D2 receptor mRNA, whereas thyroid hormone, vitamin D3, and steroid hormones (estradiol, testosterone, and corticosterone) had no effect. The transcriptional activity of the rat D2 receptor gene promoter region, which bears a retinoic acid‐responsive element, was increased by retinoic acid in transfected C6 glioma cells but not in transfected MMQ prolactin cells. Thyroid hormone and vitamin D3 were not effective in either cell line. Finally, mutations of the putative retinoic acid‐responsive element inhibited the transcriptional effect of retinoic acid. These results suggest that retinoic acid is a key factor in regulation of the embryonic onset of the dopaminergic D2 receptor.
Gene Transfer of Calbindin D28k cDNA via Herpes Simplex Virus Amplicon Vector Decreases Cytoplasmic Calcium Ion Response and Enhances Neuronal Survival Following Glutamatergic Challenge but Not Following Cyanide
Authors: Meier Timothy J. , Ho Dora Y. , Park Travis S. , Sapolsky Robert M.
Published: July 2010

Abstract: Excitatory amino acid overstimulation of neurons can lead to a marked rise in cytoplasmic Ca2+ concentration ([Ca2+])i) and be followed by neuron death from hours to days later. If the rise in [Ca2+]i is prevented, either by removing Ca2+ from the extracellular environment or by placing Ca2+ chelators in the cytosol of the stimulated cells, the neurotoxicity associated with excitotoxins can be ameliorated. We have recently shown that neurons infected with a herpes simplex virus amplicon vector expressing cDNA for calbindin D28k responded to hypoglycemia with decreased [Ca2+]i and increased survival relative to controls. We now report that vector‐infected neurons respond to glutamatergic insults with lower [Ca2+]i than controls and with increased survival. Infected neurons exposed to sodium cyanide did not respond with lower [Ca2+]i than controls, nor did they demonstrate increased survival postinsult. We examine these results in light of our earlier report and in the context of the potential of vectors like this for neuronal gene therapy.
Injury‐Related Factors and Conditions Down‐Regulate the Thrombin Receptor (PAR‐1) in a Human Neuronal Cell Line
Authors: Weinstein Jonathan R. , Lau Alice L. , Brass Lawrence F. , Cunningham Dennis D.
Published: July 2010

Abstract: Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR‐1). Mechanisms of PAR‐1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR‐1 regulation in neural cells. In the present study, PAR‐1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti‐PAR‐1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high‐affinity, specific binding to a single affinity class of receptors: KD = 3.13 and 5.25 nM, Bmax = 190.1 and 67.8 fmol/mg of protein for 125I‐ATAP2 and 125I‐SPAN12, respectively. Specificity for PAR‐1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR‐1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin‐induced PAR‐1 cleavage and internalization, with protein synthesis‐dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR‐1 expression. Significant decreases in PAR‐1 expression were induced by the protein kinase C activator phorbol 12‐myristate 13‐acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury‐related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia‐induced cell killing correlated temporally with PAR‐1 cell surface expression.
Coupling of Serotonin 5‐HT1B Receptors to Activation of Mitogen‐Activated Protein Kinase (ERK‐2) and p70 S6 Kinase Signaling Systems
Authors: Pullarkat Sajit R. , Mysels David J. , Tan Michael , Cowen Daniel S.
Published: July 2010

Abstract: Little is known about the coupling of serotonin 5‐HT1B receptors to cellular signals other than cyclic AMP. In the present studies, the activation by 5‐HT1B receptors of p70 S6 kinase and the mitogen‐activated protein kinase (MAP kinase) ERK‐2 was investigated. Studies were performed by using both nontransfected Chinese hamster ovary (CHO) cells, which express endogenous receptors at a very low density, and a stable transfected CHO cell line expressing 5‐HT1B receptors at 230 fmol/mg of membrane protein, a density similar to that expressed in cortex. In nontransfected cells, 5‐HT was found to stimulate a greater than twofold increase in MAP kinase activity with an EC50 of 20 nM. Reflecting increased density of receptors, 5‐HT caused a greater than eightfold activation of ERK‐2 in transfected cells with an EC50 of 2 nM. 5‐HT was found to also stimulate p70 S6 kinase in both nontransfected and transfected cells. The stimulation was sixfold in both types of cells, but the EC50 for 5‐HT was fourfold lower in transfected cells. The coupling of 5‐HT1B receptors to ERK‐2 and to p70 S6 kinase was inhibited by pertussis toxin, inhibitors of phosphatidylinositol 3‐kinase, and by the inhibitor of MAP kinase kinase PD098059. Activation of p70 S6 kinase, but not ERK‐2, was also inhibited by rapamycin. These findings demonstrate that 5‐HT1B receptors couple to ERK‐2 and p70 S6 kinase through overlapping, but nonidentical, pathways.
Protected‐Site Phosphorylation of Protein Kinase C in Hippocampal Long‐Term Potentiation
Authors: Sweatt J. David , Atkins Coleen M. , Johnson Joanne , English Joey D. , Roberson Erik D. , Chen Shu‐Jen , Newton Alexandra , Klann Eric
Published: July 2010

Abstract: One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long‐lasting biochemical and physiological effects in neurons. In long‐term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase‐reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the α and βII isoforms of PKC autophosphorylated in the C‐terminal domain. Using western blotting with this antiserum we observed an NMDA receptor‐mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three‐dimensional structure of the PKC catalytic core, we propose a “protected site” model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long‐lasting physiologic changes.
Modulation of [3H]Acetylcholine Release from Cultured Amacrine‐Like Neurons by Adenosine A1 Receptors
Authors: Santos Paulo F. , Santos Maria S. , Carvalho Arsélio P. , Duarte Carlos B.
Published: July 2010

Abstract: We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine‐like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N‐type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine‐like neurons, the Ca2+‐dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N‐type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX‐sensitive G proteins.
Intracellular Calcium Enhances the Ethanol Sensitivity of NMDA Receptors Through an Interaction with the C0 Domain of the NR1 Subunit
Authors: Mirshahi Tooraj , Anders Douglas L. , Ronald Kimberly M. , Woodward John J.
Published: July 2010

Abstract: Previous studies in this laboratory have shown that the ethanol inhibition of recombinant NMDA receptors expressed in Xenopus oocytes is subunit‐dependent, with the NR1/2A receptor being more sensitive than NR1/2C receptors. The ethanol sensitivity of NR1/2A receptors is reduced by substitution of the wild‐type NR1‐1a (NR1011) subunit with the calcium‐impermeable NR1 (N616R) subunit. In the present study, the ethanol inhibition of NMDA receptors was determined under different conditions to examine the role that calcium plays in determining the ethanol sensitivity of recombinant NMDA receptors. The ethanol sensitivity of NR1/2B or NR1/2C receptors was not affected by alterations in extracellular calcium levels or by coexpression with calcium‐impermeable NR1 mutants. In contrast, the inhibition of NR1/2A receptors by 100 mM ethanol was reduced in divalent‐free recording medium and was significantly increased when 10 mM calcium was used as the only charge carrier. The increase in the ethanol sensitivity of NR1/2A receptors under high‐calcium conditions was prevented by preinjection of oocytes with the calcium chelator 1,2‐bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) but not by inhibitors of calmodulin or protein kinase C. Ethanol did not alter the channel blocking activity of divalent cations on NMDA‐induced currents. The enhanced ethanol sensitivity of NR1/2A receptors in 10 mM calcium persisted when the NR1 subunit was replaced by the alternative splice variant NR1‐4a (NR1000), which lacks the C1 and C2 cassettes. However, expression of a mutant NR1 subunit that lacked the C0, C1, and C2 domains abolished the calcium‐dependent enhancement of ethanol's inhibition of NR1/2A receptors. Finally, the ethanol sensitivity of wild‐type NR1/2A receptors measured in transfected HEK 293 cells by whole cell patch‐clamp electrophysiology was significantly reduced by expression of the C‐terminal truncated NR1 subunit. These results demonstrate that the ethanol sensitivity of certain NMDA receptors is modulated by an intracellular, calcium‐dependent process that requires the C0 domain of the NR1 subunit.
Modulation of Neurosteroid Synthesis/Accumulation by l‐Ascorbic Acid in Rat Brain Tissue: Inhibition by Selected Serotonin Antagonists
Authors: Roscetti Gianna , Del Carmine Renata , Trabucchi Marco , Massotti Marino , Purdy Robert H. , Barbaccia Maria Luisa
Published: July 2010

Abstract: We have investigated the possibility that the synthesis/accumulation of neurosteroids, i.e., brain‐produced steroids putatively endowed with modulatory actions in the CNS, is regulated by monoaminergic receptor‐mediated mechanisms. In minces of rat brain cortex, l‐ascorbic acid concentration‐dependently (0.07–1.0 mM) increases the levels of pregnenolone, allotetrahydrodeoxycorticosterone, and dehydroepiandrosterone. This effect of l‐ascorbic acid is region‐dependent: in hippocampus, progesterone and allopregnanolone are also increased, whereas dehydroepiandrosterone is unchanged, and in corpus striatum only progesterone is increased significantly. 5‐Hydroxytryptamine (10 µM), 1‐(3‐chlorophenyl)piperazine (1.0 µM), and 5‐methoxytryptamine (0.4 µM) mimic the effect of l‐ascorbic acid, whereas a pretreatment with p‐chlorophenylalanine (400 mg/kg i.p., 2 days) reduces the amplitude of the l‐ascorbic acid effect on brain cortical neurosteroids. The effect of l‐ascorbic acid is blocked by the nonselective serotonin antagonists methiothepin, clozapine, methysergide, and pizotifen, but not mesulergine, spiperone, MDL 72222, and dl‐propranolol, nor by the catecholaminergic receptor antagonists prazosin and S(−)‐sulpiride. l‐Ascorbic acid is not additive with dibutyryl‐cyclic AMP and, furthermore, the inhibition of adenylate cyclase by MDL 12330A, but not of phospholipase C by U‐73122, markedly attenuates the l‐ascorbic acid‐induced increase of pregnenolone in rat brain cortical minces. Together these data suggest that l‐ascorbic acid plays a role in the modulation of neurosteroidogenesis, presumably by favoring the activation of the purported serotonin type 6 receptor by endogenous serotonin.
Regulation of the Ca2+ Sensitivity of Exocytosis by Rab3a
Authors: Johannes Ludger , Lledo Pierre‐Marie , Chameau Pascal , Vincent Jean‐Didier , Henry Jean‐Pierre , Darchen François
Published: July 2010

Abstract: Ca2+ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP‐binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca2+]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca2+ dialysis through a patch‐clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2–4 µM) [Ca2+], but did not change the maximal activity observed at 10 µM free [Ca2+]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense‐injected cells dialyzed with 10 µM [Ca2+] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca2+ dependence of exocytosis and that its activity is modulated further in a stimulus‐dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.
Iron Uptake in Blood‐Brain Barrier Endothelial Cells Cultured in Iron‐Depleted and Iron‐Enriched Media
Authors: Van Gelder W. , Huijskes‐Heins M. I. E. , Cleton‐Soeteman M. I. , Van Dijk J. P. , Van Eijk H. G.
Published: July 2010

Abstract: Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood‐brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood‐brain barrier endothelial cells were grown in either iron‐enriched or iron‐depleted medium. Iron‐enriched cells showed a reduction in surface‐bound and total transferrin receptor numbers compared with iron‐depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron‐enriched cultures was higher, whereas the transferrin receptor externalization rate in iron‐enriched cultures was lower than the rate in iron‐depleted cultures. Moreover, blood‐brain barrier endothelial cells cultured in iron‐enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood‐brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.
The Polarity of Choroid Plexus Epithelial Cells In Vitro Is Improved in Serum‐Free Medium
Authors: Hakvoort Ansgar , Haselbach Matthias , Wegener Joachim , Hoheisel Dirk , Galla Hans‐Joachim
Published: July 2010

Abstract: The influence of culture conditions on the development of normal characteristics of the choroid plexus epithelium has been investigated in vitro with respect to polarity, barrier properties, transport, and secretory activity. Withdrawal of serum supplement in the culture medium of cells grown on filters caused morphologically visible changes by an increased trimming of microvilli at the apical membrane side, which is accompanied by an increased expression of the Na+,K+‐ATPase. Moreover cells under serum‐free conditions exhibit structural changes in tight junctional zonula occludens protein‐1 (ZO‐1) organization, a reduced permeability, and a drastically increased electrical resistance from 150 Ω· cm2 in the presence of serum to 1,500 Ω· cm2 after serum withdrawal. Under these conditions, cell monolayers are able to build up a transcellular proton gradient and to secrete fluid into the upper (apical) filter compartment, which is accompanied by a polarized secretion of proteins like transthyretin. Active transport of the dyes fluorescein and phenol red by the organic anion transporter is found to be driven by the Na+,K+‐ATPase. We come to the conclusion that removal of serum favors the differentiation process of the plexus epithelium in vitro, which brings the cell culture model closer to the physiological situation in vivo. We present preliminary evidence that epidermal growth factor may be one component in serum preventing the proper in vitro differentiation.
Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro
Authors: Tilling Thomas , Korte Dorothea , Hoheisel Dirk , Galla Hans‐Joachim
Published: July 2010

Abstract: The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TER) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one‐to‐one mixtures of these proteins. Moreover, a one‐to‐one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (∼350 Ω× cm2) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one‐to‐one combinations of these proteins elevated transcellular resistances significantly (2.3‐ to 2.9‐fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (∼1,000 Ω× cm2) could, by contrast, be increased only 1.1‐ to 1.2‐fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood‐brain barrier.
Energy Dependency of Glucocorticoid Exacerbation of gp120 Neurotoxicity
Authors: Brooke Sheila M. , Howard Sarah A. , Sapolsky Robert M.
Published: July 2010

Abstract: The HIV envelope glycoprotein, gp120, a well documented neurotoxin, may be involved in AIDS‐related dementia complex. gp120 works through an NMDA receptor‐ and calcium‐dependent mechanism to damage neurons. We have previously demonstrated that both natural and synthetic glucocorticoids (GCs) exacerbate gp120‐induced neurotoxicity and calcium mobilization in hippocampal mixed cultures. GCs, steroid hormones secreted during stress, are now shown to work in conjunction with gp120 to decrease ATP levels and to work synergistically with gp120 to decrease the mitochondrial potential in hippocampal cultures. Furthermore, energy supplementation blocked the ability of GCs to worsen gp120's effects on neuronal survival and calcium mobilization. A GC‐induced reduction in glucose transport in hippocampal neurons, as previously documented, may contribute to this energetic dependency. These results may have clinical significance, considering the common treatment of severe cases of Pneumocystis carinii pneumonia, typical of HIV infection, with large doses of synthetic GCs.
Prolonged Expression of Interferon‐Inducible Protein‐10 in Ischemic Cortex After Permanent Occlusion of the Middle Cerebral Artery in Rat
Authors: Wang Xinkang , Ellison Julie A. , Siren Anna‐Leena , Lysko Paul G. , Yue Tian‐Li , Barone Frank C. , Shatzman Allan , Feuerstein Giora Z.
Published: July 2010

Abstract: Focal cerebral ischemia elicits local inflammatory reaction as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain. Interferon‐inducible protein‐10 (IP‐10) is a member of the C‐X‐C chemokine family that possesses potent chemoattractant actions for monocytes, T cells, and smooth muscle cells. To investigate a potential role of IP‐10 in focal stroke, we studied the temporal expression of IP‐10 mRNA after occlusion of the middle cerebral artery in rat by means of northern analysis. IP‐10 mRNA expression after focal stroke demonstrated a unique biphasic profile, with a marked increase early at 3 h (4.9‐fold over control; p < 0.01), a peak level at 6 h (14.5‐fold; p < 0.001) after occlusion of the middle cerebral artery, and a second wave induction 10–15 days after ischemic injury (7.2‐ and 9.3‐fold increase for 10 and 15 days, respectively; p < 0.001). In situ hybridization confirmed the induced expression of IP‐10 mRNA and revealed its spatial distribution after focal stroke. Immunohistochemical studies demonstrated the expression of IP‐10 peptide in neurons (3–12 h) and astroglial cells (6 h to 15 days) of the ischemic zone. To explore further the potential role of IP‐10 in focal stroke, we demonstrated a dose‐dependent chemotactic action of IP‐10 on C6 glial cells and enhanced attachment of rat cerebellar granule neurons. Taken together, the data suggest that ischemia induces IP‐10, which may play a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neuron attachment/sprouting after focal stroke.
Elevated “Hydroxyl Radical” Generation In Vivo in an Animal Model of Amyotrophic Lateral Sclerosis
Authors: Bogdanov Mikhail B. , Ramos Louis E. , Xu Zuoshang , Beal M. Flint
Published: July 2010

Abstract: Mutations in the enzyme copper/zinc superoxide dismutase‐1 (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS). The means by which the mutations cause FALS appears to be due to an adverse property of the mutant SOD1 protein that may involve increased generation of free radicals. We used in vivo microdialysis to measure the conversion of 4‐hydroxybenzoic acid to 3,4‐dihydroxybenzoic acid (3,4‐DHBA) as a measure of “hydroxyl radical‐like” production in transgenic amyotrophic lateral sclerosis (ALS) mice with the G93A mutation as well as littermate controls. The conversion of 4‐hydroxybenzoic acid to 3,4‐DHBA was significantly increased in the striatum of transgenic ALS mice at baseline but not in mice overexpressing wild‐type human SOD1. Following administration of 3‐nitropropionic acid 3,4‐DHBA generation was significantly increased as compared with baseline, and the increase in the transgenic ALS mice was significantly greater than those in controls, whereas the increase in mice overexpressing wild‐type human SOD1 was significantly attenuated. The present results provide in vivo evidence that expression of mutations in SOD1 can lead to increased generation of “hydroxyl radical‐like” activity, which further implicates oxidative damage in the pathogenesis of ALS.