bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of Glutamate Transporter Subtypes During Human Brain Development
Authors: Bar‐Peled Osnat , Ben‐Hur Herzel , Biegon Anat , Groner Yoram , Dewhurst Steve , Furuta Akiko , Rothstein Jeffrey D.
Published: July 2010

Abstract: In the mature brain, removal of glutamate from the synaptic cleft plays an important role in the maintenance of subtoxic levels of glutamate. This requirement is handled by a family of glutamate transporters, EAAT1, EAAT2, EAAT3, and EAAT4. Due to the involvement of glutamate also in neuronal development, it is believed that glutamate transport plays a role in developmental processes as well. Therefore, we have used immunohistochemical and immunoblot analysis to determine the distribution of the four glutamate transporters during human brain development using human pre‐ and postnatal brain tissue. Regional analysis showed that each transporter subtype has a unique distribution during development. EAAT2 was the most prominent glutamate transporter subtype and was highly enriched in cortex, basal ganglia, cerebellum, and thalamus in all ages examined. EAAT1 immunoreactivity was lower than that of EAAT2, with predominant localization in cortex, basal ganglia, hippocampus, and periventricular region. EAAT3 was located mainly in cortex, basal ganglia, and hippocampus, and EAAT4 was found only in cortex, hippocampus, and cerebellar cortex. The distinct regional distribution of various EAAT subtypes and also the transient expression of specific EAAT subtypes during development suggest multiple functional roles for glutamate transporters in the developing brain.
Agonist Action at D2(short) Dopamine Receptors Determined in Ligand Binding and Functional Assays
Authors: Gardner B. R. , Hall D. A. , Strange P. G.
Published: July 2010

Abstract: Mechanisms of agonist action at the G protein‐coupled D2(short) dopamine receptor expressed in Chinese hamster ovary cells have been investigated. Agonist binding was assayed in the presence and absence of GTP (100 µM). Data in the absence of GTP were fitted best by a two‐site model (apomorphine, dopamine, 10,11‐dihydroxy‐N‐n‐propylnorapomorphine hydrochloride, and quinpirole) or a one‐site model [bromocriptine, dihydroergocristine, and (−)‐3‐(3‐hydroxyphenyl)‐N‐propylpiperidine hydrochloride], whereas in the presence of GTP a one‐site model was the best fit for all compounds. Agonist binding parameters were used to provide a measure of the ability of the agonist to stabilise the ternary complex of agonist/receptor/G protein. Agonist stimulation of [35S]guanosine 5′‐O‐(3‐thiotriphosphate) ([35S]‐GTPγS) binding for a range of agonist concentrations was measured and the EC50 and maximal effects determined. The initial rates of [35S]GTPγS binding induced by maximally stimulating agonist concentrations were also recorded. Simultaneous inhibition of agonist‐stimulated [35S]GTPγS binding and receptor occupancy by spiperone was determined. Agonist inhibition of forskolin‐stimulated cyclic AMP accumulation was determined for a range of agonist concentrations and the EC50 and maximal inhibition recorded. The data on the maximal agonist responses showed that it was possible to detect a spectrum of agonist efficacy (partial and full agonism) in both functional assays. The data on the apparent potencies of agonists to elicit the functional responses showed that different extents of amplification of response were seen for different agonists in both assays. The maximal activity data have been compared with the stabilisation of the agonist/receptor/G protein ternary complex as measured in binding assays.
Tetracycline‐Regulated Transgene Expression in Hippocampal Neurones Following Transfection with Adenoviral Vectors
Authors: Harding T. C. , Geddes B. J. , Noel J. D. , Murphy D. , Uney J. B.
Published: July 2010

Abstract: A transfer system that enabled the efficient introduction of transgenes into neurones and the quantitative control of the expressed transgene would greatly facilitate studies into neuronal gene function. To develop such a system we incorporated the tetracycline (Tet)‐responsive On/Off regulatory elements into type‐5 adenoviral (Ad) vectors. Regulation of transgene expression following transfection was measured by placing the enhanced green fluorescent protein (EGFP) gene upstream of the Tet regulatory element. The results showed that cultures of primary hippocampal cells could be transfected with very high efficiency (<70%) by the AdTet‐On and AdTet‐Off systems. Following transfection with the AdTet‐On system no EGFP‐fluorescent cells could be detected until doxycycline was added. The AdTet‐Off system showed the reverse transcriptional regulation, in that the addition of Tet caused EGFP fluorescence to be abolished.
Effect of Docosahexaenoic Acid on the Synthesis of Phosphatidylserine in Rat Brain Microsomes and C6 Glioma Cells
Authors: Garcia Martha C. , Ward Glenn , Ma Yee‐Chung , Salem Norman , Kim Hee‐Yong
Published: July 2010

Abstract: Docosahexaenoic acid (22:6n‐3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n‐3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n‐3‐deficient diet showed a dramatic reduction of 22:6n‐3 content (1.7 ± 0.1%) when compared with control animals (15.0 ± 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n‐6) content, which replaced the 22:6n‐3 phospholipids with 22:5n‐6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n‐3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1‐stearoyl‐2‐docosapentanoyl (18:0/22:5n‐6) species, particularly in phosphatidylcholine. Incorporation of [3H]serine into PS in rat brain microsomes from n‐3‐deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n‐3‐supplemented media (10–40 µM) showed a significant increase in the synthesis of [3H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n‐3 may be a modulator of PS synthesis.
Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro
Authors: Hackam Abigail S. , Wang Tian‐Li , Guggino William B. , Cutting Garry R.
Published: July 2010

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA‐gated chloride channel receptors. GABAA receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABAC receptors appear to contain ρ subunits. However, retinal cells exhibiting GABAC responses express α, β, and ρ subunits, raising the possibility that GABAC receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABAA receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full‐length ρ1, ρ2, or the N‐terminal domain of ρ1 that contains signals for ρ‐subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild‐type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full‐length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABAA subunits into GABAC and GABAA receptors, respectively.
Lysophosphatidic Acid Induces Necrosis and Apoptosis in Hippocampal Neurons
Authors: Holtsberg Frederick W. , Steiner Marion R. , Keller Jeffrey N. , Mark Robert J. , Mattson Mark P. , Steiner Sheldon M.
Published: July 2010

Abstract: A diverse body of evidence indicates a role for the lipid biomediator lysophosphatidic acid (LPA) in the CNS. This study identifies and characterizes the induction of neuronal death by LPA. Treatment of cultured hippocampal neurons from embryonic rat brains with 50 µM LPA resulted in neuronal necrosis, as determined morphologically and by the release of lactate dehydrogenase. A concentration of LPA as low as 10 µM led to the release of lactate dehydrogenase. In contrast, treatment of neurons with 0.1 or 1.0 µM LPA resulted in apoptosis, as determined by chromatin condensation. In addition, neuronal death induced by 1 µM LPA was characterized as apoptotic on the basis of terminal dUTP nick end‐labeling (TUNEL) staining, externalization of phosphatidylserine, and protection against chromatin condensation, TUNEL staining, and phosphatidylserine externalization by treatment with N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone, a broad‐spectrum inhibitor of caspases, i.e., members of the interleukin‐1β converting enzyme family. Studies with antagonists of ionotropic glutamate receptors did not indicate a significant role for these receptors in apoptosis induced by 1 µM LPA. LPA (1 µM) also induced a decrease in mitochondrial membrane potential. Moreover, pretreatment of neurons with cyclosporin A protected against the LPA‐induced decrease in mitochondrial membrane potential and neuronal apoptosis. Thus, LPA, at pathophysiological levels, can induce neuronal apoptosis and could thereby participate in neurodegenerative disorders.
Cytosolic Calmodulin Is Increased in SK‐N‐SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores
Authors: McGinnis K. M. , Shariat‐Madar Z. , Gnegy M. E.
Published: July 2010

Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK‐N‐SH. Increasing the intracellular Ca2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca2+ pools in the muscarinic receptor‐mediated increases in cytosolic CaM in SK‐N‐SH cells. Stimulus‐mediated changes in intracellular Ca2+ were monitored in fura‐2‐loaded cells, and CaM was measured by radioimmunoassay in the 100,000‐g cytosol and membrane fractions. The influx of extracellular Ca2+ normally seen with carbachol treatment in SK‐N‐SH cells was eliminated by pretreatment with the nonspecific Ca2+ channel blocker Ni2+. Blocking the influx of extracellular Ca2+ had no effect on carbachol‐mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca2+ from the medium did not affect carbachol‐mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol‐mediated increase of intracellular free Ca2+ by pretreatment with the cell‐permeant Ca2+ chelator BAPTA/AM did attenuate the carbachol‐mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca2+ into the cell by K+ depolarization was assessed. Incubation of SK‐N‐SH cells with 60 mM K+ elicited an immediate and persistent increase in intracellular free Ca2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca2+, which releases Ca2+ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K+ depolarization. These data demonstrate that release of Ca2+ from the intracellular store is important for the carbachol‐mediated redistribution of CaM in human neuroblastoma SK‐N‐SH cells.
Cerebrovascular Accumulation and Increased Blood‐Brain Barrier Permeability to Circulating Alzheimer's Amyloid β Peptide in Aged Squirrel Monkey with Cerebral Amyloid Angiopathy
Authors: Mackic Jasmina B. , Weiss Martin H. , Miao Wesley , Kirkman Erlinda , Ghiso Jorge , Calero Miguel , Bading James , Frangione Blas , Zlokovic Berislav V.
Published: July 2010

Abstract: Senescent squirrel monkey is a valuable model to study pathogenesis of cerebrovascular amyloid angiopathy (CAA). Cerebrovascular sequestration and blood‐brain barrier (BBB) permeability to 125I‐amyloid β(1‐40) synthetic peptide (sAβ1‐40) were studied in adult versus aged squirrel monkey 1 h after a single intravenous injection. In aged monkey, the half‐time of elimination of sAβ1‐40, te1/2, was prolonged by 0.6 h, the systemic clearance, ClSS, was reduced from 1.8 to 1.1 ml/min/kg, and the mean residence time of intact peptide in the circulation was increased by 1 h (45%). In adult monkey, cerebrovascular sequestration of intact sAβ1‐40 was significant, and the BBB permeability was 18.6‐fold higher than for inulin. In aged monkey, the sequestration of intact sAβ1‐40 by cortical and leptomeningeal microvessels and the BBB permeability were increased by 5.9, 1.8‐, and 2.1‐fold, respectively, in the presence of an unchanged barrier to inulin. In brain parenchyma of aged animals, 76.1% of circulating sAβ1‐40 remained intact versus 45.7% in adult. We conclude that multiple age‐related systemic effects, i.e., reduced body elimination and systemic clearance of sAβ1‐40, and reduced peripheral metabolism, may act in concert with BBB mechanisms, i.e., increased transendothelial transport and microvascular accumulation of blood‐borne sAβ1‐40, and reduced brain metabolism to enhance the development of CAA.
Effect of Isolation‐Rearing on Conditioned Dopamine Release In Vivo in the Nucleus Accumbens of the Rat
Authors: Fulford Allison J. , Marsden Charles A.
Published: July 2010

Abstract: Intracerebral microdialysis in conjunction with HPLC coupled to electrochemical detection was used to investigate the effect of isolation‐rearing in the rat on extracellular dopamine (DA) and its metabolites in vivo, in the shell region of the nucleus accumbens, in response to footshock and in relation to a conditioned emotional response. Male Lister hooded rats were reared from weaning for 6–8 weeks in either social isolation or groups of five. In the training phase, rats were exposed to a novel environment for 10 min where they experienced mild footshock. Footshock caused an immediate increase in basal extracellular DA levels in both rearing groups relative to control rats. However, the increase in extracellular DA was prolonged in the case of the isolation‐reared rats and significantly greater than in group‐reared rats. Exposure to the novel environment without shock (control groups) did not significantly alter basal extracellular DA in the nucleus accumbens shell; 140 min later rats were returned to the testing box (contextual stimulus) without receiving footshock. The contextual stimulus increased basal extracellular DA in the nucleus accumbens of both groups of rats with respect to controls; however, this increase was significantly greater and more prolonged in isolates. Extracellular levels of the metabolites 3,4‐dihydroxyphenylacetic acid and homovanillic acid did not differ between isolation‐ and group‐reared rats, and they were not significantly affected by either footshock or the contextual stimulus. These results suggest that exposure to footshock and a contextual stimulus are associated with increases in basal extracellular DA levels in the nucleus accumbens shell. The results also support evidence in favour of an isolation‐induced enhancement in dopaminergic activity in the nucleus accumbens, which probably underlies aspects of the behavioural syndrome associated with isolation.
Imaging the Brain Marijuana Receptor: Development of a Radioligand that Binds to Cannabinoid CB1 Receptors In Vivo
Authors: Gatley S. John , Lan Ruoxi , Volkow Nora D. , Pappas Naomi , King Payton , Wong Christopher T. , Gifford Andrew N. , Pyatt Beatrice , Dewey Stephen L. , Makriyannis Alexandros
Published: July 2010

Abstract: The major active ingredient of marijuana, (−)‐Δ9‐tetrahydrocannabinol, exerts its psychoactive effects via binding to cannabinoid CB1 receptors, which are widely distributed in the brain. Radionuclide imaging of CB1 receptors in living human subjects would help explore the presently unknown physiological roles of this receptor system, as well as the neurochemical consequences of marijuana dependence. Currently available cannabinoid receptor radioligands are exceedingly lipophilic and unsuitable for in vivo use. We report the development of a novel radioligand, [123I]AM281{N‐(morpholin‐4‐yl)‐5‐(4‐[123I]iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide}, that is structurally related to the CB1‐selective antagonist SR141716A [N‐(piperidin‐1‐yl)‐5‐(4‐chlorophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide]. Baboon single photon emission computed tomography studies, mouse brain dissection studies, and ex vivo autoradiography in rat brain demonstrated rapid passage of [123I]AM281 into the brain after intravenous injection, appropriate regional brain specificity of binding, and reduction of binding after treatment with SR141716A. AM281 has an affinity in the low nanomolar range for cerebellar binding sites labeled with [3H]SR141716A in vitro, and binding of [123I]AM281 is inhibited by several structurally distinct cannabinoid receptor ligands. We conclude that [123I]AM281 has appropriate properties for in vivo studies of cannabinoid CB1 receptors and is suitable for imaging these receptors in the living human brain.
Peroxynitrite and Hydrogen Peroxide Induced Cell Death in the NSC34 Neuroblastoma × Spinal Cord Cell Line: Role of Poly(ADP‐Ribose) Polymerase
Authors: Cookson Mark R. , Ince Paul G. , Shaw Pamela J.
Published: July 2010

Abstract: The reaction of superoxide and nitric oxide results in the formation of peroxynitrite, a long lived and highly reactive oxidant species. It has been suggested that the formation of peroxynitrite in vivo may contribute to cell death in some neurological conditions. We have examined the effect of peroxynitrite on cell death in the NSC34 spinal cord cell line. A brief (30 min) exposure to either peroxynitrite or hydrogen peroxide caused delayed cell death with an EC50 for both of ∼1 mM. Cell death was prevented by the RNA synthesis inhibitor actinomycin D and included DNA damage as an early event. We sought to clarify the potential role of the DNA binding enzyme poly(ADP‐ribose) polymerase (PARP) in cell death in these cells. Several PARP inhibitors [benzamide, 3‐aminobenzamide, nicotinamide, and 6(5H)‐phenanthridinone] prevented cell death, but the inactive analogue benzoic acid did not. However, there was no evidence of cleavage of PARP, which occurs in apoptosis via the activation of the caspase CPP32. Therefore, we suggest that PARP contributes to neuronal injury as an early event, probably by lethal NAD depletion, without any requirement for proteolytic cleavage.
Regulation of Glial Cell Line‐Derived Neurotrophic Factor Release from Rat C6 Glioblastoma Cells
Authors: Verity A. Neil , Wyatt T. L. , Hajos B. , Eglen R. M. , Baecker P. A. , Johnson R. M.
Published: July 2010

Abstract: We have monitored glial cell line‐derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24‐h treatment with fibroblast growth factor‐1, −2, or −9 elevated secreted GDNF protein levels five‐ to 10‐fold. The proinflammatory cytokines interleukin‐1β, interleukin‐6, tumor necrosis factor‐α, and lipopolysaccharide elevated GDNF release 1.5‐ to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13‐didecanoate (10 nM) activation of protein kinase C, the Ca2+ ionophore A23187 (1 µM), okadaic acid (10 nM) inhibition of type‐2A protein phosphatases, nitric oxide donors (1 mM), and H2O2 (1 mM)‐induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 µM) or dibutyryl cyclic AMP (1 mM) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 µM). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.
Evidence for Neuronal Origin and Metabotropic Receptor‐Mediated Regulation of Extracellular Glutamate and Aspartate in Rat Striatum In Vivo Following Electrical Stimulation of the Prefrontal Cortex
Authors: Lada Mark W. , Vickroy Thomas W. , Kennedy Robert T.
Published: July 2010

Abstract: Extracellular levels of glutamate (Glu) and aspartate (Asp) were measured at 5‐s intervals in the striatum of chloral hydrate‐anesthetized rats by using microdialysis coupled to an automated assay system based on capillary electrophoresis with laser‐induced fluorescence. Application of a single 10‐s train of depolarizing pulses to the prefrontal cortex caused a rapid increase in Glu and Asp concentrations (200–300% of basal value), which returned to basal level within 60 s. The stimulated rise in Glu and Asp concentrations was blocked completely by 2 µM tetrodotoxin or depletion of extracellular Ca2+, suggesting a neuronal origin of the Glu and Asp. Infusion of the Glu transport inhibitor l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (200 µM) increased resting Glu and Asp levels by 300–500% without altering electrically stimulated changes in Glu and Asp concentration. Stimulated Glu and Asp concentration changes were suppressed by 91 and 73%, respectively, by the metabotropic Glu receptor agonist (1S,3R)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylate (200 µM). This effect was blocked by the metabotropic Glu receptor antagonist (RS)‐α‐methylcarboxyphenylglycine (MCPG; 200 µM). MCPG alone produced no effect on electrically stimulated changes in Glu and Asp levels; however, in the presence of l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid, MCPG produced a five‐ to sixfold increase in stimulated overflow. Based on these results, it is concluded that release of Glu and Asp from corticostriatal neurons can be inhibited by activation of metabotropic Glu autoreceptors, which may be an important determinant of excitatory transmission at striatal synapses.
κ Receptor Activation Attenuates l‐trans‐Pyrrolidine‐2,4‐Dicarboxylic Acid‐Evoked Glutamate Levels in the Striatum
Authors: Rawls Scott M. , McGinty Jacqueline F.
Published: July 2010

Abstract: The effects of local κ receptor activation and blockade on extracellular striatal glutamate levels evoked by reverse microdialysis of l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (l‐trans‐PDC) were investigated. l‐trans‐PDC elevates extracellular glutamate levels in vivo by acting as a competitive substrate for plasma membrane excitatory amino acid transporters. The selective κ‐opioid receptor agonist U‐69593 (1‐100 nM) significantly attenuated l‐trans‐PDC‐stimulated glutamate levels in a concentration‐dependent manner. The selective κ receptor antagonist nor‐binaltorphimine (1‐100 nM) reversed the U‐69593‐induced decrease in l‐trans‐PDC‐evoked glutamate levels also in a concentration‐dependent manner, indicating that the U‐69593‐induced reduction was mediated by κ receptor activation. In addition, nor‐binaltorphimine significantly elevated basal extracellular glutamate levels, implying that κ receptors tonically regulate glutamate efflux in the striatum. Previous data from this laboratory have shown that l‐trans‐PDC‐evoked extracellular glutamate levels are partially calcium‐sensitive. The present study demonstrated that the inhibition of l‐trans‐PDC‐evoked glutamate levels by reduced calcium perfusion was not altered by U‐69593. Therefore, κ receptors regulate the calcium‐dependent component of l‐trans‐PDC‐evoked extracellular glutamate levels in the striatum.
Spinal Neurokinin NK1 Receptor Down‐Regulation and Antinociception: Effects of Spinal NK1 Receptor Antisense Oligonucleotides and NK1 Receptor Occupancy
Authors: Hua Xiao‐Ying , Chen Ping , Polgar Erika , Nagy Istvan , Marsala Martin , Phillips Elsa , Wollaston Laura , Urban Laszlo , Yaksh Tony L. , Webb Michael
Published: July 2010

Abstract: To define the effects of antisense oligonucleotides on spinal neurokinin 1 (NK1) receptor function in nociceptive processing, several antisense oligonucleotides directed against the NK1 receptor mRNA were intrathecally injected into rats via an implanted catheter, and their effect on the behavioural response to formalin injected into the paw was assessed. We observed that there was no significant reduction of pain behaviour or immunostaining of spinal NK1 receptors after repeated daily intrathecal treatment with an antisense oligonucleotide. However, spinal application of substance P (SP) in the antisense oligonucleotide‐treated animals resulted in a profound and long‐lasting reduction in the behavioural response to formalin injection, and a parallel reduction in the NK1 receptor immunoreactivity normally observed in spinal dorsal horn. Intrathecal SP in the control groups, i.e., rats treated with an oligonucleotide containing four mismatched bases, the corresponding sense oligonucleotide, a mixture of the sense and the antisense oligonucleotides, in each case had no effect. The effects of SP were blocked by NK1 receptor antagonists and were not mimicked by NMDA. The mechanism underlying these effects is not clear. It may be due to partial degradation of the internalised receptors, which cannot be replaced by newly synthesised receptors because of the action of the NK1 antisense oligonucleotide.
CuZn‐Superoxide Dismutase, Extracellular Superoxide Dismutase, and Glutathione Peroxidase in Blood from Individuals Homozygous for Asp90Ala CuZn‐Superoxide Dismutase Mutation
Authors: Andersen P. M. , Nilsson P. , Forsgren L. , Marklund S. L.
Published: July 2010

Abstract: The Asp90Ala CuZn‐superoxide dismutase mutation is associated with amyotrophic lateral sclerosis (ALS) in both homo‐ and heterozygous form. We analyzed antioxidant enzymes in blood from 44 individuals homozygous and 114 individuals heterozygous for the Asp90Ala mutation as well as 66 blood relatives carrying the wild‐type allele only. Erythrocyte CuZn‐superoxide dismutase activity was reduced by 9% in the homozygous individuals, confirming previous findings on a smaller cohort. The specific activity of Asp90Ala mutant CuZn‐superoxide dismutase in erythrocytes was equal to that of isolated mutant enzyme and slightly higher than that of isolated wild‐type enzyme. There was no evidence for the presence of inactive mutant molecules in erythrocytes, and the lower activity is due to the occurrence of fewer active molecules. There were no significant differences between the groups in plasma extracellular superoxide dismutase content, and the erythrocyte glutathione peroxidase activities were virtually identical. Also, there were no differences in these parameters between homozygous individuals with or without ALS. There was no evidence for any association with ALS of a polymorphic extracellular superoxide dismutase mutation, Arg213Gly. The absence of response of the blood antioxidant enzymes to the Asp90Ala CuZn‐superoxide dismutase mutation does not support the theory that the ALS‐linked CuZn‐superoxide dismutase mutations cause disease by increased oxidant stress.
Proteolytic Degradation of Alzheimer's Disease Amyloid β‐Peptide by a Metalloproteinase from Microglia Cells
Authors: Mentlein Rolf , Ludwig Ralf , Martensen Ilka
Published: July 2010

Abstract: The cerebral deposition of amyloid β‐peptide (Aβ) is a histopathological characteristic of Alzheimer's disease. Because an impaired clearance of Aβ might be involved in the disease, we investigated the proteolytic degradation of synthetic Aβ (40‐residue peptide) in cultures of glial cells and characterized a protease involved. Whereas rat astrocytes had a very low degradation capacity, cultivated rat microglia cells cleaved Aβ. Microglia activity was considerably enhanced by stimulation with lipopolysaccharide and to a lesser extent by phorbol esters. Most of the Aβ‐degrading activity was released into the medium. By use of selective inhibitors the protease was characterized as a metalloprotease of ∼200 kDa that was different from neutral endopeptidase (a neuropeptide‐degrading enzyme), matrix metalloproteases, or macrophage elastase. Its activity was efficiently reduced by four hydroxamic acid‐based zinc‐metalloprotease inhibitors that have been shown to inhibit membrane protein secretases (disintegrins). We conclude that activated microglia cells might impair amyloid plaque formation by release of a metalloprotease that degrades soluble Aβ before polymerization.
Novel Endogenous 1,2,3,4‐Tetrahydroisoquinoline Derivatives: Uptake by Dopamine Transporter and Activity to Induce Parkinsonism
Authors: Kawai Hiroshi , Makino Yukiko , Hirobe Masaaki , Ohta Shigeru
Published: July 2010

Abstract: We designed as candidate metabolites and synthesized two 1‐benzyl‐1,2,3,4‐tetrahydroisoquinoline derivatives containing a dopamine moiety: 1‐(3′,4′‐dihydroxybenzyl)‐1,2,3,4‐tetrahydroisoquinoline (3′,4′‐DHBnTIQ) and 1‐benzyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline (6,7DHBnTIQ). Both were detected in mouse brain as endogenous amines by gas chromatography/mass spectrometry. 3′,4′DHBnTIQ induced parkinsonism in mice when chronically administered intraperitoneally, whereas 6,7DHBnTIQ did not despite the structural similarity of the two compounds. This difference may be related to cellular uptake: In rat striatal synaptosomes, these compounds were intracellularly transported by the dopamine transporter with Km values of 6.14 and 7.82 µM and Vmax values of 214.3 and 112.2 pmol/min/mg of protein, respectively. Thus, endogenous 3′,4′DHBnTIQ may be actively transported into dopaminergic neurons and accumulated there, contributing at least in part to the induction of idiopathic Parkinson's disease.
Exocytotic and Nonexocytotic Modes of Glutamate Release from Cultured Cerebellar Granule Cells During Chemical Ischaemia
Authors: Pocock Jennifer M. , Nicholls David G.
Published: July 2010

Abstract: The mechanism of glutamate release from cultured cerebellar granule neurones in response to a chemical model of ischaemia (10 mM 2‐deoxyglucose plus 1 mM sodium cyanide) was investigated. In the first 2 min of ischaemia, release of preloaded d‐[3H]aspartate could be extensively attenuated by tetanus toxin and bafilomycin A1 and was dependent on the activation of Ca2+ channels sensitive to the “Q” type Ca2+ channel antagonist, ω‐conotoxin‐MVIIC. During this period, ATP/ADP ratios fell rapidly. The extent of release in the first 2 min was comparable to that evoked by 2‐min depolarization by 50 mM KCl. Free Ca2+ concentrations, determined in neurites and somata, did not increase until after 2 min. The neurite increase in cellular Ca2+ precedes that of the cell somata. Release of d‐[3H]aspartate was partially inhibited by the NMDA receptor antagonist MK‐801, which also delayed the increase in free Ca2+ concentration. Prolonging the period of ischaemia to 6 and 10 min produced no further increase in the apparently exocytotic component of release, but initiated an extensive nonexocytotic release of the amino acid. Studies with the synaptic vesicle membrane probe FM1‐43 in which released amino acid was removed by superfusion indicated that Ca2+‐dependent exocytosis was delayed in this system. It is concluded that chemical ischaemia initiates an initial exocytotic followed by nonexocytotic release and that the former is facilitated by NMDA receptor activation. These events occur in cells that are still able to exclude propidium iodide, indicating that cell death has not yet occurred.
Homophilic Binding Properties of Galectin‐3: Involvement of the Carbohydrate Recognition Domain
Authors: Kuklinski Stephan , Probstmeier Rainer
Published: July 2010

Abstract: Galectin‐3, an animal lectin specific for β‐galactosides, is composed of three different domains. The N‐terminal half of the molecule (N domain) consists of a short N‐terminal segment followed by glycine‐, proline‐, and tyrosine‐rich tandem repeats. The C‐terminal domain (C domain) harbors the carbohydrate recognition domain homologous to other members of the galectin family of lectins. Galectin‐3 aggregates in solution, and participation of the N domain of the molecule in this process has already been demonstrated. Using a solid‐phase radioligand binding assay, which allows the direct analysis of galectin‐3 self‐association, here we provide evidence that the carbohydrate recognition domain of the lectin is involved in carbohydrate‐dependent homophilic interactions: (a) Radiolabeled galectin‐3 binds to immobilized galectin‐3, and the addition of unlabeled galectin‐3 in solution increases the rate of binding of radiolabeled lectin; (b) binding of radiolabeled galectin‐3 to immobilized galectin‐3 is inhibited by the C domain; (c) binding of radiolabeled galectin‐3 to immobilized galectin‐3 or the C domain is inhibited by lactose but not by sucrose; and (d) the radiolabeled C domain does not bind to immobilized C domain. Taken together, these data suggest that in addition to the N domain, the homophilic interactions of galectin‐3 are mediated by the C domain.
A “Septide‐Sensitive” Receptor Is Not Involved in Tachykinin‐Mediated Secretory and Inositol Phosphate Responses in Rat Parotid Gland: Are Several Transduction Pathways Involved After the Stimulation of the NK1 Receptor?
Authors: Huleux Claire , Berthier Annabel , Rossignol Bernard , Dreux Catherine
Published: July 2010

Abstract: In the rat parotid gland, the neuropeptide substance P (SP), as well as SP(4–11), and septide elicited inositol phosphate production (EC50 values 0.44, 2, and 20 nM, respectively). No additivity of the maximal response to the three agonists was observed. SP, SP(4–11), and septide also stimulated protein secretion; for SP, two EC50 were determined (0.5 and 160 nM), whereas a single one could be determined for SP(4–11) and septide (EC50 values 15 and 20 nM, respectively). The selective tachykinin NK1 receptor antagonist RP67580 acted as a competitive inhibitor of both SP‐ and SP(4–11)‐induced inositol phosphate production. Its effect on septide‐induced inositol phosphate production was noncompetitive. RP67580 is apparently as potent at antagonizing septide, SP, or SP(4–11) (in all cases KB = 3 nM). These results show that in parotid gland, only NK1 receptors are activated by SP, SP(4–11), and septide. We also showed that the protein secretion stimulated by SP was inhibited competitively by RP67580, whereas the effect of RP67580 was noncompetitive on protein secretion when SP(4–11) or septide was used. Our data indicate that in rat parotid gland, the existence of a specific “septide‐sensitive” receptor can be ruled out and that only the NK1 receptor is present and mediates cellular responses. Taken together, these results show that in this tissue the NK1 receptor would present at least two different binding sites that could be coupled to different transduction pathways and that would regulate protein secretion.
GAP‐43 Augmentation of G Protein‐Mediated Signal Transduction Is Regulated by Both Phosphorylation and Palmitoylation
Authors: Nakamura Fumio , Strittmatter Philipp , Strittmatter Stephen M.
Published: July 2010

Abstract: The neuronal protein GAP‐43 is concentrated at the growth cone membrane, where it is thought to amplify the signal transduction process. As a model for its neuronal effects, GAP‐43 protein injection into Xenopus laevis oocytes strongly augments the calcium‐sensitive chloride current evoked by the G protein‐coupled receptor stimulation. We have now examined a series of GAP‐43 mutants in this system and determined those regions of GAP‐43 required for this increase in current flux. As expected, palmitoylation inhibits signal amplification in oocytes by blocking G protein activation. Unexpectedly, a second domain of GAP‐43 (residues 35–50) containing a protein kinase C phosphorylation site at residue 41 is also necessary for augmentation of G protein‐coupled signals in oocytes. This region is not required for activation of isolated Go but is necessary for GAP‐43 binding to isolated calmodulin and to isolated protein kinase C. Substitution of Asp for Ser41 inactivates GAP‐43 as a signal facilitator in oocytes. This mutation blocks GAP‐43 binding to both protein kinase C and calmodulin. Thus, GAP‐43 regulates an oocyte signaling cascade via coordinated, simultaneous G protein activation and interaction with either calmodulin or protein kinase C.
Involvement of Platelet‐Activating Factor in Cell Death Induced Under Ischemia/Postischemia‐Like Conditions in an Immortalized Hippocampal Cell Line
Authors: Shi Leng‐Chu , Wang Hoau‐Yan , Friedman Eitan
Published: July 2010

Abstract: The involvement of platelet‐activating factor (PAF) in cell damage induced by ischemia/postischemia‐like conditions was studied in a hippocampus‐derived cell line, HN33.11. Cells exposed to N2‐saturated glucose‐free HEPES‐buffered saline (ischemia) for 5 h followed by 18 h of incubation in serum‐free control medium (postischemia reincubation) remained 67.4 ± 2.4% viable in comparison with sham‐treated cells. Analysis of DNA fragmentation in combination with Hoechst 33258 staining indicates that apoptosis is the dominant mode of cell death in the present model. PAF level during 10 h of ischemia was unchanged. However, an increase in PAF accumulation was found early during the reincubation period that followed 5 h of ischemia. Peak PAF concentrations were noted at 2 h after initiation of reincubation and rapidly declined to control level after 7 h of reincubation. Consistent with a role of PAF in mediating cell death under ischemia/postischemia reincubation in this model, the PAF antagonist BN 50739 exerted a dose‐dependent protective effect. Maximal protection (85.7 ± 5.4%) of the cells from ischemia/reincubation‐induced cell damage was achieved at 0.1 µM BN 50739. The PAF antagonist lacked any protective effect against ischemia‐induced cell death. On the other hand, the addition of the stable PAF analogue 1‐O‐hexadecyl‐2‐N‐methylcarbamyl‐sn‐glycero‐3‐phosphocholine (MC‐PAF) at the onset of ischemia potentiated ischemia/reincubation‐induced apoptosis—an effect that was blocked by BN 50739. Pretreatment of HN33.11 cells with the Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N,N‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM) also provided a protective effect against ischemia/reincubation‐induced cell damage. BAPTA‐AM increased cell viability by 50%. Pretreatment with BAPTA‐AM also decreased ischemia/reincubation‐induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in part mediating apoptosis under ischemia/postischemia‐like conditions in HN33.11 cells.
Calcium Regulation of Agonist Binding to α7‐Type Nicotinic Acetylcholine Receptors in Adult and Fetal Rat Hippocampus
Authors: Ospina Jose A. , Broide Ron S. , Acevedo Diana , Robertson Richard T. , Leslie Frances M.
Published: July 2010

Abstract: Quantitative autoradiography was used to compare the binding properties of α7‐type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of 125I‐α‐bungarotoxin (125I‐α‐BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of 125I‐α‐BTX binding, as well as α‐cobratoxin and nicotine potency to displace 125I‐α‐BTX, did not change with age. Addition of Ca2+ to the assay buffer did not alter 125I‐α‐BTX binding, or α‐cobratoxin inhibition of 125I‐α‐BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca2+ on agonist affinity was dose‐dependent, with an EC50 value of 0.25–0.5 mM. Ca2+ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal 125I‐α‐BTX binding sites are largely similar across age. Ca2+ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the α7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.
Differential Inhibition by α‐Conotoxin‐MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices
Authors: Kaiser S. A. , Soliakov L. , Harvey S. C. , Luetje C. W. , Wonnacott S.
Published: July 2010

Abstract: The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α‐conotoxin‐MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)‐anatoxin‐a‐evoked release of [3H]dopamine from rat striatal synaptosomes and slices. α‐Conotoxin‐MII (112 nM) completely blocked acetylcholine‐evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC50 = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα‐conotoxin‐MII. On perfused striatal synaptosomes and slices, α‐conotoxin‐MII dose‐dependently inhibited [3H]dopamine release evoked by 1 µM (±)‐anatoxin‐a with IC50 values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose‐response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α‐conotoxin‐MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2‐like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)‐anatoxin‐a‐evoked [3H]dopamine release by α‐conotoxin‐MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α‐conotoxin‐MII‐insensitive nAChR.
Dopaminergic Regulation of Striatal Acetylcholine Release: The Critical Role of Acetylcholinesterase Inhibition
Authors: Acquas E. , Fibiger H. C.
Published: July 2010

Abstract: This study examined the effects of different levels of acetylcholinesterase (AChE) inhibition on dopaminergic regulation of striatal acetylcholine (ACh) release as estimated by in vivo brain microdialysis. Systemic administration of d‐amphetamine (2 or 10 mg/kg) increased the striatal output of ACh when the AChE inhibitor neostigmine (0.1 µM) was present in the perfusion fluid. In contrast, when the same experiments were conducted at 0.01 µM neostigmine, d‐amphetamine failed to affect (2 mg/kg) or significantly decreased (10 mg/kg) striatal ACh output. The inhibitory action of the D2 receptor agonist quinpirole (0.2 mg/kg) was significantly greater at 0.01 µM than at 0.1 µM neostigmine. Similarly, there was a nonsignificant trend for the D2 antagonist raclopride (1 mg/kg) to stimulate ACh release to a greater extent at the low neostigmine concentration. In contrast, the stimulant effects of systemic administration of the D1 agonist A‐77636 (1.46 mg/kg) on striatal ACh release were the same at the two neostigmine concentrations. These results demonstrate that the concentration of an AChE inhibitor in the perfusion solution can quantitatively and even qualitatively influence the manner in which dopaminergic agents regulate ACh overflow in the striatum. On comparing the present results with earlier reports concerning the effects of d‐amphetamine on tissue concentrations of ACh, it is tentatively concluded that a low neostigmine concentration is the more physiologically relevant condition. Under such conditions, at moderate doses d‐amphetamine does not appear to alter striatal ACh release, with this likely being due to the opposing actions of D1 and D2 receptors. Nevertheless, until the endogenous interstitial concentrations of striatal ACh can be measured by other methods, the physiological relevance of ACh microdialysis studies in the striatum will remain uncertain.
High Calcium Permeability of Serotonin 5‐HT3 Receptors on Presynaptic Nerve Terminals from Rat Striatum
Authors: Rondé Philippe , Nichols Robert A.
Published: July 2010

Abstract: The serotonin 5‐HT3 receptor, a ligand‐gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5‐HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5‐HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5‐HT3 receptor‐induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1‐(m‐chlorophenyl)biguanide (mCPBG), a highly specific 5‐HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG‐induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5‐HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N‐methyl‐d‐glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage‐gated Ca2+ channel blockade by 10 µM Co2+/Cd2+ or by 1 µMω‐conotoxin MVIIC/1 µMω‐conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5‐HT3 receptor activation in NG108‐15 cells by 1 µM mCPBG was substantially reduced by 10 µM Co2+/Cd2+ and was completely blocked by 1 µM nitrendipine, an L‐type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5‐HT3 receptors, presynaptic 5‐HT3 receptors appear to be uniquely calcium‐permeant.
[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies
Authors: Fredholm Bertil B. , Lindström Karin , Dionisotti Silvio , Ongini Ennio
Published: July 2010

Abstract: We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3H]CGS 21680) or A1 receptors (N6‐[3H]cyclohexyladenosine), it was found that SCH 58261 is close to 800‐fold selective for rat brain A2A versus A1 receptors (Ki values of 1.2 nM versus 0.8 µM). Moreover, receptor autoradiography showed that [3H]SCH 58261, in concentrations below 2 nM, binds only to the dopamine‐rich regions of the rat brain, with a KD value of 1.4 (0.8–1.8) nM. The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 nM, the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3H]SCH 58261 with the following estimated Ki values (nM): 2‐hex‐1‐ynyl‐5′‐N‐ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N6‐cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µM) or Mg2+ (10 mM). The present results show that in its tritium‐labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3H]CGS 21680.