bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrogen Peroxide Induces a Long‐Lasting Inhibition of the Ca2+‐Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+
Authors: Zoccarato Franco , Valente Marina , Alexandre Adolfo
Published: July 2010

Abstract: We studied the action of H2O2 on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H2O2 (50–150 µM) for a few minutes results in a long‐lasting depression of the Ca2+‐dependent exocytosis of glutamate, induced by KCl or by the K+‐channel inhibitor 4‐aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H2O2, although a transient decrease was observed after the treatment. H2O2 did not promote peroxidation, as judged by the formation of malondialdehyde. In indo‐1‐loaded synaptosomes, the treatment with H2O2 did not modify significantly the KCl‐induced increase of [Ca2+]i. H2O2 inhibited exocytosis also when the latter was induced by increasing [Ca2+]i with the Ca2+ ionophore ionomycin. The effects of H2O2 were unchanged in the presence of superoxide dismutase and the presence of the Fe3+ chelator deferoxamine. These results appear to indicate that H2O2, apparently without damaging the synaptosomes, induces a long‐lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.
Involvement of Different Types of Voltage‐Sensitive Calcium Channels in the Presynaptic Regulation of Noradrenaline Release in Rat Brain Cortex and Hippocampus
Authors: Sabrià Josefa , Pastor Conxita , Clos Maria Victoria , Garcia Alicia , Badia Albert
Published: July 2010

Abstract: Transmitter release at the nerve terminal is mediated by the influx of Ca2+ through voltage‐sensitive calcium channels (VSCCs). Many types of VSCCs have been found in neurons (T, N, L, and P), but uncertainty remains about which ones are involved in neuronal excitation‐secretion coupling. Specific ligands for the L‐ and N‐type VSCCs were used to determine which of these subtypes might be involved in the K+‐evoked [3H]noradrenaline release from superfused rat brain cortical and hippocampal synaptosomes. In cortical presynaptic terminals the 1,4‐dihydropyridine agonist Bay K 8644 enhanced the K+ (15 mM)‐evoked [3H]noradrenaline release. This effect was reversed by the 1,4‐dihydropyridine antagonists nimodipine and nitrendipine. The L‐type VSCC ligands had no effect on hippocampal synaptosomes. In contrast, the N‐type VSCC blocker ω‐conotoxin markedly reduced the K+‐evoked [3H]noradrenaline release in nerve terminals from both regions. Inhibition was greater in hippocampal synaptosomes. When applied together the inhibitory actions of nimodipine and ω‐conotoxin were approximately additive. These findings indicate that both L‐ and N‐type VSCCs participate in noradrenaline release in rat brain cortex and suggest that noradrenergic terminals in the two regions examined may have distinct populations of VSCCs: L type in cortex and N type in hippocampus.
Characterisation of Antibodies Specific for Chick Brain Neural Cell Adhesion Molecules Which Cause Amnesia for a Passive Avoidance Task
Authors: Mileusnic Radmila , Rose Steven P. R. , Lancashire Chris , Bullock Sarah
Published: July 2010

Abstract: Antisera were prepared against six postsynaptic density glycoprotein fractions (150–180, 62–80, 50, 41, 33, and 28 kDa) that show enhanced fucosylation during memory formation after training day‐old chicks in a one‐trial passive avoidance task. Each antiserum was tested for its possible effect on memory retention. Bilateral intracranial injections of two of the antisera, R‐1 and R‐6, or their IgGs (IgG‐1 and IgG‐6), resulted in amnesia for the passive avoidance task when chicks were tested 24 h later. IgG‐1 and IgG‐6 antibodies were amnestic only when injected 5.5 h after training, and had no effect when injections were made 30 min before training, thus resembling an effect previously observed with polyclonal or monoclonal anti‐N‐CAM antibodies. IgG‐1 and IgG‐6 antibodies were found to be specific for protein epitopes of glycoproteins that contain a high amount of N‐linked mannose and fucose, and a very low amount of polysialic acid and O‐linked galactose. Absorption of IgG‐6 antibodies with neural cell adhesion molecule (N‐CAM) isolated from synaptic plasma membranes derived from day‐old chick brain resulted in loss of amnestic effect. As we have previously shown that long‐term memory for the passive avoidance task requires two waves of glycoprotein synthesis, the first occurring immediately after training and the second 5–8 h later, the present results suggest strongly that isoforms of N‐CAM molecules with a low level of sialic acid are involved specifically in the establishment of an enduring memory for the experience of the passive avoidance task in chicks, possibly by stabilising changes in synaptic connectivity that encode the memory.
Dopamine‐Derived 1‐Methyl‐6,7‐Dihydroxyisoquinolines as Hydroxyl Radical Promoters and Scavengers in the Rat Brain: In Vivo and In Vitro Studies
Authors: Maruyama Wakako , Dostert Philippe , Naoi Makoto
Published: July 2010

Abstract: The effects of derivatives of dopamine‐derived isoquinoline, (R)‐1‐methyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline [or (R)‐salsolinol] on hydroxyl radical production were studied in vivo and in vitro. As reported previously, (R)‐salsolinol is N‐methylated in the brain into N‐methyl‐(R)‐salsolinol, which is further oxidized into the 1,2‐dimethyl‐6,7‐dihydroxyisoquinolinium ion. Using in vivo microdialysis, we measured hydroxyl radical levels in the rat striatum by HPLC after derivatization to 2,3‐dihydroxybenzoic acid with salicylic acid. (R)‐Salsolinol and the isoquinolinium ion (40 and 200 µM) and N‐methyl‐(R)‐salsolinol (200 µM) reduced in vivo radical formation, with reduction of dopamine catabolism. (R)‐Salsolinol and the isoquinolinium ion reduced in vitro hydroxyl radical production from dopamine autoxidation. On the other hand, 40 µMN‐methyl‐(R)‐salsolinol increased the hydroxyl radical level in the striatum, and the radical production by its autoxidation was confirmed in vitro. N‐Methyl‐(R)‐salsolinol affected neither in vivo dopamine catabolism nor in vitro production of hydroxyl radicals from dopamine. These results show that (R)‐salsolinol and N‐methyl‐(R)‐salsolinol may be neuroprotective and neurotoxic, respectively, and thus might be involved in the pathogenesis of Parkinson's disease.
Participation of Noradrenergic Neurotransmission in the Enhancement of Baroreceptor Reflex Response by Substance P at the Nucleus Tractus Solitarii of the Rat: A Reverse Microdialysis Study
Authors: Chan Julie Y. H. , Tsou Mei‐Yung , Len Wen‐Bin , Lee Tak‐Yu , Chan Samuel H. H.
Published: July 2010

Abstract: We applied reverse microdialysis and HPLC analysis to evaluate the participation of noradrenergic neurotransmission in modulation of the baroreceptor reflex response by substance P at the nucleus tractus solitarii in Sprague‐Dawley rats anesthetized with pentobarbital sodium (50 mg/kg, i.p., with 20 mg/kg/h.i.v. supplement). Continuous infusion of substance P (600 µM) at 1 µl/min into the nucleus tractus solitarii through a stereo‐taxically positioned microdialysis probe (active exchange length, 180–200 µm; diameter, 220 µm) for 1 h elicited an enhancement of the baroreceptor reflex response. This facilitatory effect correlated positively, during the 60‐min infusion period, with the time course of increase in the extracellular concentration of substance P and noradrenaline in the nucleus tractus solitarii. Experimentally elevating the concentration of noradrenaline at this medullary nucleus also augmented the baroreceptor reflex sensitivity. On the other hand, depletion of the noradrenergic fibers and nerve terminals at the nucleus tractus solitarii with DSP4 diminished the enhancement of baroreceptor reflex response and the corresponding elevation in extracellular concentration of noradrenaline by substance P. Microinfusion of noradrenaline into the nucleus tractus solitarii in DSP4‐treated animals, however, potentiated the baroreceptor reflex response. These results suggest that the enhancement of baroreceptor reflex response by substance P may involve an increase in the concentration of noradrenaline at the nucleus tractus solitarii via a presynaptic mechanism.
Differential Effects of Chloral Hydrate and Pentobarbital Sodium on a Cocaine Level and Its Catecholamine Response in the Medial Prefrontal Cortex: A Comparison with Conscious Rats
Authors: Pan Wynn H. T. , Lai Yu‐Jun , Chen Nien‐Hsien
Published: July 2010

Abstract: Adult male Sprague‐Dawley rats anesthetized with chloral hydrate and pentobarbital sodium were used as two different treatment groups. Conscious rats were used as a control group. By using baseline (precocaine) concentration as 100%, after cocaine administration (3.0 mg/kg i.v.), the maximal dopamine (DA) increase occurring at the first microdialysis collection period (20 min) in the medial prefrontal cortex was 299 ± 46% for the chloral hydrate group, 168 ± 12% for the pentobarbital sodium group, and 325 ± 23% for the conscious group. At the same time, norepinephrine (NA) increases reached a maximum and were 162 ± 20%, 100 ± 5%, and 141 ± 17%, respectively. The maximal changes of DA and NA in the chloral hydrate group and in the control group were both significantly higher than that in the pentobarbital sodium group. Meanwhile, the cocaine concentration was higher over a 100‐min period of time in the chloral hydrate group when compared with the pentobarbital group and the control group. The peak cocaine concentration in dialysate occurred in the same time slot of maximal DA and NA responses, which were 0.65 ± 0.08, 0.30 ± 0.02, and 0.41 ± 0.05 µM, respectively. Anesthetics suppress the pharmacologic response of neurons, which may explain the difference in catecholamine response between the pentobarbital sodium and the conscious groups. Conversely, because there was no significant difference in DA and NA response between the chloral hydrate group and the conscious group, it may possibly be due to the balancing effect between the higher existing cocaine concentration and the anesthetic suppression on pharmacological response of neurons in the chloral hydrate group. The effect of guide cannula implantation on the cocaine‐induced catecholamine response was also evaluated.
Quantification of Different Transferrin Receptor Pools in Primary Cultures of Porcine Blood‐Brain Barrier Endothelial Cells
Authors: Van Gelder W. , Huijskes‐Heins M. I. E. , Van Dijk J. P. , Cleton‐Soeteman M. I. , Van Eijk H. G.
Published: July 2010

Abstract: Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood‐brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood‐brain barrier endothelial cells, we separately quantified surface‐bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large “inactive” intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100‐fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve‐fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.
Characterisation of the Binding of [3H]WAY‐100635, a Novel 5‐Hydroxytryptamine1A Receptor Antagonist, to Rat Brain
Authors: Khawaja Xavier , Evans Non , Reilly Yvonne , Ennis Christine , Minchin Michael C. W.
Published: July 2010

Abstract: The specific binding of [3H]WAY‐100635 {N‐[2‐[4‐(2‐[O‐methyl‐3H]methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐(2‐pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [3H]WAY‐100635 association (k+1 = 0.069 ± 0.015 nM−1 min−1) and dissociation (k−1 = 0.023 ± 0.001 min−1) followed monoexponential kinetics. Saturation binding isotherms of [3H]WAY‐100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (Bmax) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [3H]WAY‐100635 was ∼36% higher compared with that of 8‐hydroxy‐2‐(di‐n‐[3H]‐propylamino)tetralin ([3H]8‐OH‐DPAT). The binding affinity of [3H]WAY‐100635 was significantly lowered by the divalent cations CaCl2 (2.5‐fold; p < 0.02) and MnCl2 (3.6‐fold; p < 0.05), with no effect on Bmax. Guanyl nucleotides failed to influence the KD and Bmax parameters of [3H]WAY‐100635 binding to 5‐HT1A receptors. The pharmacological binding profile of [3H]WAY‐100635 was closely correlated with that of [3H]8‐OH‐DPAT, which is consistent with the labelling of 5‐hydroxytryptamine1A (5‐HT1A) sites in rat hippocampus. [3H]WAY‐100635 competition curves with 5‐HT1A agonists and partial agonists were best resolved into high‐ and low‐affinity binding components, whereas antagonists were best described by a one‐site binding model. In the presence of 50 µM guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5‐HT1A receptor. However, a residual (16 ± 2%) high‐affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP‐insensitive sites.
Analysis of S100A1 Expression During Skeletal Muscle and Neuronal Cell Differentiation
Authors: Zimmer Danna B. , Landar Aimee
Published: July 2010

Abstract: To better understand the mechanisms that regulate the function of the calcium‐binding proteins S100A1 and S100B in developing systems, we have examined the level of, subcellular distribution of, and target proteins for these proteins in skeletal muscle (L6S4) and neuronal (PC12) cell lines. Both undifferentiated and differentiated L6 and PC12 cells express S100A1 and not S100B. Whereas S100A1 protein levels were higher in differentiated cells than in undifferentiated cells, steady‐state mRNA levels did not change in differentiated L6 cells and decreased in differentiated PC12 cells when compared with undifferentiated cells. These results suggest that posttranscriptional rather than transcriptional mechanisms are responsible for increased S100A1 protein expression in myotubes and neurons. The colocalization of S100A1 staining with wheat germ agglutinin staining suggests that S100A1 is associated with the Golgi apparatus and secretory vesicles in PC12 and L6 cells. Using a gel overlay technique, S100A1‐binding proteins were detected in undifferentiated and differentiated PC12 and L6 cells and the patterns observed were similar to those observed in brain and skeletal muscle, respectively. Although changes in the intensity of some binding proteins were detected, the overall pattern did not change when differentiated and undifferentiated cells were compared. These results suggest that the complement of S100A1‐binding proteins does not change during differentiation, only the levels of some binding proteins. Altogether, our data demonstrate that the L6 and PC12 cell lines are excellent in vitro model systems for studying S100A1 expression and mechanisms that regulate S100A1 expression, subcellular distribution, and interaction with target proteins.
Glial‐Neuronal Interactions as Studied by Cerebral Metabolism of [2‐13C]Acetate and [1‐13C]Glucose: An Ex Vivo 13C NMR Spectroscopic Study
Authors: Hassel Bjørnar , Sonnewald Ursula , Fonnum Frode
Published: July 2010

Abstract: Mice were injected intravenously with [2‐13C]‐acetate or [1‐13C]glucose and killed after 5, 15, or 30 min. Another group of animals was injected three times subcutaneously during 30 min with [2‐13C]acetate to achieve a steady‐state‐like situation. Brain extracts were analyzed by 13C NMR spectroscopy, and the percent enrichment of various carbon positions was calculated for amino acids, lactate, and glucose. Results obtained with [2‐13C]acetate, which is metabolized by glia and not by neurons, showed that glutamine originated from a glial tricarboxylic acid cycle (TCA cycle) that loses 65% of its intermediates per turn of the cycle. This TCA cycle was associated with pyruvate carboxylation, which may replenish virtually all of this loss, as seen from the labeling of glutamine from [1‐13C]glucose. From the C‐3/C‐4 labeling ratios in glutamine and glutamate and from the corresponding C‐3/C‐2 labeling ratio in GABA obtained with [2‐13C]acetate, it was concluded that the carbon skeleton of glutamine to some extent was passed through TCA cycles before glutamate and GABA were formed. Thus, astrocytically derived glutamine is not only a precursor for transmitter amino acids but is also an energy substrate for neurons in vivo. Furthermore, the neuronal TCA cycles may be control points in the synthesis of transmitter amino acids. Injection of [2‐13C]acetate led to a higher 13C enrichment of the C‐2 in glutamate and of the corresponding C‐4 in GABA than in the C‐3 of either compound. This could reflect cleavage of [2‐13C]‐citrate and formation of [3‐13C]oxaloacetate and acetyl‐CoA, i.e., the first step in fatty acid synthesis. [3‐13C]‐Oxaloacetate would, after entry into a TCA cycle, give the observed labeling of glutamate and GABA.
Comparison of (R)‐[3H] Tomoxetine and (R/S)‐[3H] Nisoxetine Binding in Rat Brain
Authors: Gehlert Donald R. , Schober Douglas A. , Gackenheimer Susan L.
Published: July 2010

Abstract: (R)‐[3H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower‐affinity site. The goal of the present study was to identify the nature of this low‐affinity site by comparing the binding properties of (R)‐[3H]tomoxetine with those of (R/S)‐[3H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas (R)‐[3H]tomoxetine also bound to a second, lower‐affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE‐containing neurons. However, low levels of (R)‐[3H]‐tomoxetine binding were seen in the caudate‐putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where (R/S)‐[3H]nisoxetine binding was almost absent. In homogenates of the caudate‐putamen, the NE uptake inhibitors desipramine and (R)‐nisoxetine and the serotonin (5‐HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 nM (R)‐nisoxetine to mask the binding of (R)‐[3H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [3H]citalopram. Therefore, (R)‐[3H]tomoxetine binds to the NE uptake site with high affinity and the 5‐HT uptake site with somewhat lower affinity.
Monoaminergic Effects of Folinic Acid, l‐DOPA, and 5‐Hydroxytryptophan in Dihydropteridine Reductase Deficiency
Authors: Goldstein David S. , Hahn Si‐Houn , Holmes Courtney , Tifft Cynthia , Harvey‐White Judith , Milstein Sheldon , Kaufman Seymour
Published: July 2010

Abstract: Plasma and CSF concentrations of endogenous l‐DOPA, catecholamines, and metabolites of monoamines were assayed in a patient with atypical phenylketonuria due to absent dihydropteridine reductase (DHPR), before and during treatment with folinic acid, Sinemet, and 5‐hydroxytryptophan. The patient had low but detectable levels of l‐DOPA, 3,4‐dihydroxyphenylacetic acid (DOPAC), and 3,4‐dihydroxyphenylglycol (DHPG) in plasma and low but detectable levels of these compounds and of homovanillic acid (HVA) and 5‐hydroxyindoleacetic acid (5‐HIAA) in CSF, with approximately normal plasma and CSF levels of norepinephrine [noradrenaline (NA)]. Folinic acid treatment approximately doubled plasma levels of l‐DOPA, NA, DOPAC, and DHPG, compared with values during dietary phenylalanine restriction alone. Detection of l‐DOPA, catecholamines, and monoamine metabolites in this patient indicates that monoamine synthesis in humans does not absolutely require DHPR. The results are consistent with the existence of an alternative biochemical pathway, with folinic acid treatment augmenting activity along this pathway. Low plasma levels of l‐DOPA, DOPAC, and DHPG may reflect decreased catecholamine synthesis and turnover in sympathetic nerves, with compensatory increases in exocytotic release normalizing plasma NA levels.
Immunocytochemical Analysis of Axonal Outgrowth in Synaptotagmin Mutations
Authors: Littleton J. Troy , Upton Lindy , Kania Artur
Published: July 2010

Abstract: Synaptotagmin is a synaptic vesicle specific protein that binds calcium and phospholipids in vitro and is required for calcium‐regulated fusion of synaptic vesicles with the presynaptic membrane. We have examined the possible requirement for synaptotagmin in axonal outgrowth by following neuronal development in Drosophila embryos deficient for the synaptotagmin gene. We find that synaptotagmin is expressed abundantly in axons and growth cones before synapse formation in wild‐type embryos. Using antibodies to the intravesicular domain of synaptotagmin to label live embryos, we demonstrate that vesicle populations containing synaptotagmin actively undergo exocytosis during axonogenesis. We have used immunocytochemical techniques to examine the distribution of the axonal protein Fasciclin II, the presynaptic membrane protein syntaxin, and the synaptic vesicle protein cysteine string protein, in synaptotagmin null mutations. The distribution of these proteins is similar in wild‐type and synaptotagmin mutant embryos, suggesting that synaptotagmin is not required for axonogenesis in the CNS or PNS. Based on these findings, we suggest that the molecular mechanisms underlying vesicular‐mediated membrane expansion during axonal outgrowth are distinct from those required for synaptic vesicle fusion during neurotransmitter release.
L1 and N‐CAM Antibodies Trigger Protein Phosphatase Activity in Growth Cone‐Enriched Membranes
Authors: Klinz Stephan G. , Schachner Melitta , Maness Patricia F.
Published: July 2010

Abstract: Triggering of the cell adhesion molecules L1 or N‐CAM in a nerve growth cone membrane fraction from fetal rat brain with purified L1 or N‐CAM or specific antibodies decreases the steady‐state levels of protein tyrosine phosphorylation in the membranes. Here we report that triggering of L1 and N‐CAM in the growth cone‐enriched membrane fraction with a subset of antibodies directed against the extracellular region of L1 and N‐CAM elicited dephosphorylation of endogenous protein substrates, indicating the presence of a cell adhesion molecule‐activated phosphatase. The most prominent substrates were a membrane‐associated 200‐kDa protein and tubulin, both of which were dephosphorylated on tyrosine and serine/threonine residues in response to L1 or N‐CAM triggering. The antibody‐induced phosphatase was inhibited by agents that blocked tyrosine and serine/threonine phosphatases, including sodium orthovanadate, vanadyl sulfate, zinc cations, heparin, and sodium pyrophosphate. Purified L1 and N‐CAM fragments and other antibodies reacting with the extracellular region of these adhesion molecules did not activate the phosphatase but did inhibit tyrosine phosphorylation. These properties suggested that triggering of L1 and N‐CAM can lead to either phosphatase activation or tyrosine kinase inhibition in growth cone membranes. These findings implicate protein phosphatases in addition to tyrosine kinases as components of L1 and N‐CAM intracellular signaling pathways in growth cones.
Allosteric Interactions Among Agonists and Antagonists at 5‐Hydroxytryptamine3 Receptors
Authors: Bonhaus Douglas W. , Stefanich Eric , Loury Dana N. , Hsu Sherry A. O. , Eglen Richard M. , Wong Erik H. F.
Published: July 2010

Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5‐hydroxytryptamine3 (5‐HT3) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5‐HT3 receptors. The affinities of competitive 5‐HT3 receptor antagonists were similar regardless of whether the receptors were labeled with [3H]RS‐42358, [3H]granisetron, or 1‐(m‐[3H]chlorophenyl)biguanide ([3H]mCPG). By contrast, the affinities of the agonists 5‐HT, mCPG, and phenylbiguanide were approximately 10‐fold higher when the receptors were labeled with [3H]mCPG. The dissociation of [3H]mCPG, [3H]RS‐42358, and [3H]RS‐25259, but not [3H]granisetron, from both cloned and native 5‐HT3 receptors was markedly slower in the presence of 5‐HT or 2‐methyl‐5‐HT than in the presence of antagonists such as RS‐42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5‐HT3 receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.
Local Influence of Endogenous Norepinephrine on Extracellular Dopamine in Rat Medial Prefrontal Cortex
Authors: Gresch Paul J. , Sved Alan F. , Zigmond Michael J. , Finlay Janet M.
Published: July 2010

Abstract: Noradrenergic and dopaminergic projections converge in the medial prefrontal cortex and there is evidence of an interaction between dopamine (DA) and norepinephrine (NE) terminals in this region. We have examined the influence of drugs known to alter extracellular NE on extracellular NE and DA in medial prefrontal cortex using in vivo microdialysis. Local application of the NE uptake inhibitor desipramine (1.0 µM) delivered through a microdialysis probe increased extracellular DA (+149%) as well as NE (+201%) in medial prefrontal cortex. Furthermore, desipramine potentiated the tail shock‐induced increase in both extracellular DA (stress alone, +64%; stress + desipramine, +584%) and NE (stress alone, +55%; stress + desipramine, +443%). In contrast, local application of desipramine did not affect extracellular DA in striatum, indicating that this drug does not influence DA efflux directly. Local application of the α2‐adrenoceptor antagonist idazoxan (0.1 or 5.0 mM) increased extracellular NE and DA in medial prefrontal cortex. Conversely, the α2‐adrenoceptor agonist clonidine (0.2 mg/kg; i.p.) decreased extracellular NE and DA in medial prefrontal cortex. These results support the hypothesis that NE terminals in medial prefrontal cortex regulate extracellular DA in this region. This regulation may be achieved by mechanisms involving an action of NE on receptors that regulate DA release (heteroreceptor regulation) and/or transport of DA into noradrenergic terminals (heterotransporter regulation).
Phospholipase A2 Down‐Regulates the Affinity of [3H]AMPA Binding to Rat Cortical Membranes
Authors: Dev Kumlesh K. , Honoré Tage , Henley Jeremy M.
Published: July 2010

Abstract: The effects of exogenous phospholipase A2 on the binding of α‐[3H]amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate ([3H]AMPA) to rat cortical membranes in the presence of the chaotrope potassium thiocyanate were assessed. Pretreatment of membranes with secretory phospholipase A2 (sPLA2) elicited a concentration‐dependent decrease in specific [3H]AMPA binding due mainly to a decrease in affinity (KD). This observation, together with protease inhibitor and western blot evidence, suggest that the sPLA2 effect is not due to proteolysis. The sPLA2‐evoked decrease was temperature and calcium dependent. Inclusion of the specific inhibitor oleoyloxyethyl phosphocholine or preincubation of the enzyme with reducing agents to degrade its secondary structure significantly reduced the sPLA2 inhibition. These results suggest that the effects of sPLA2 arise from an enzymatic action rather than a competitive interaction at the AMPA binding site. However, arachidonic acid, a major metabolite of sPLA2 action, did not cause a similar decrease in the affinity of [3H]AMPA binding. In contrast to the effects on [3H]AMPA binding, sPLA2 caused an increase in [3H]CNQX binding, which is in accordance with the functionality of the AMPA receptor complex. These results suggest that sPLA2 may play a role in the physiological and pathophysiological regulation of AMPA receptors.
The Intracellular Component of Cellular 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction Is Specifically Inhibited by β‐Amyloid Peptides
Authors: Shearman Mark S. , Hawtin Stuart R. , Tailor Vipula J.
Published: July 2010

Abstract: In vitro cell culture model systems for investigating the biochemical mechanisms involved in the neurodegenerative actions of β‐amyloid peptide (β‐AP) have been established. Using rat pheochromocytoma PC12 or human epitheloid HeLa cell lines, submicromolar concentrations of the β‐AP fragments β1–40, β1–39, and β25–35, but not β1–28, were found to inhibit the reduction of the redox dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). In both cell lines, the β‐AP‐sensitive component represented ∼70% of total cellular MTT reduction. When the reduction of a series of structurally related dyes was compared with that of MTT, the reduction of 3α‐naphthyl‐2‐phenyl‐5‐(4‐nitrophenyl)‐2H‐tetrazolium chloride (NTV) was also found to be sensitive to β25–35, but that of seven other redox dyes was not. A property common to MTT and NTV is that they are both readily taken up into PC12 and HeLa cells and do not require an artificial electron coupling agent to be reduced. Microscopic analysis of MTT‐formazan product formation in PC12 and HeLa cells following β25–35 treatment revealed that it was the intracellular component of the reduction of this dye that was abolished. These results support the hypothesis that the cellular reduction of MTT represents a specific indicator of the initial events underlying the mechanism of β‐AP toxicity.
Effect of a Novel Prolyl Endopeptidase Inhibitor, JTP‐4819, on Prolyl Endopeptidase Activity and Substance P‐ and Arginine‐Vasopressin‐Like Immunoreactivity in the Brains of Aged Rats
Authors: Toide Katsuo , Okamiya Kazuhiro , Iwamoto Yohko , Kato Takeshi
Published: July 2010

Abstract: The effects of a novel prolyl endopeptidase (PEP) inhibitor, (S)‐2‐[[(S)‐2‐(hydroxyacetyl)‐1‐pyrrolidinyl]carbonyl]‐N‐(phenylmethyl)‐1‐pyrrolidinecarboxamide (JTP‐4819), on the PEP activity in the brain and on the contents of substance P (SP)‐ and arginine‐vasopressin (AVP)‐like immunoreactivity (LI) in the cerebral cortex and hippocampus of young and aged rats were investigated using enzyme immunoassay. JTP‐4819 exhibited a concentration‐dependent in vitro inhibitory action on PEP activity in the brains of both young and aged rats, with IC50 values of ∼0.7 and 0.8 nM, respectively. A single dose of JTP‐4819 (3 mg/kg, p.o.) increased the SPLI content in the cerebral cortex but not the hippocampus of aged rats (23–24 months old). In addition, repeated administration of JTP‐4819 (1 mg/kg, p.o., for 21 days) increased the SPLI content in the cerebral cortex and restored the SPLI content in the hippocampus, which had decreased with aging. In contrast, single (1 mg/kg, p.o.) and repeated (1 mg/kg, p.o., for 21 days) administration of JTP‐4819 only tended to increase the AVPLI content of the hippocampus and cerebral cortex in aged rats, respectively. These results indicate that JTP‐4819 increases the cerebral and hippocampal SPLI content in aged rats by inhibiting the action of PEP.
Influence of Perfusate Glucose Concentration on Dialysate Lactate, Pyruvate, Aspartate, and Glutamate Levels Under Basal and Hypoxic Conditions: A Microdialysis Study in Rat Brain
Authors: Ronne‐Engström Elisabeth , Carlson Hans , Yansheng Liu , Ungerstedt Urban , Hillered Lars
Published: July 2010

Abstract: The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 mM glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no‐net‐flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant differences in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose‐free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.
Resiniferatoxin‐Amide and Analogues as Ligands for Protein Kinase C and Vanilloid Receptors and Determination of Their Biological Activities as Vanilloids
Authors: Acs Geza , Lee Jeewoo , Marquez Victor E. , Wang Shaomeng , Milne George W. A. , Du Linh , Lewin Nancy E. , Blumberg Peter M.
Published: July 2010

Abstract: The naturally occurring diterpene resiniferatoxin (RTX) is an ultrapotent analogue of capsaicin. Acting on polymodal afferent neurons, RTX induces a generally similar pattern of responses as does capsaicin. However, the two compounds, as well as other vanilloid derivatives, display different relative potencies for different responses. In the present study, we examined the vanilloid‐like activities of two new derivatives, the amide analogue of RTX and phorbol 12,13‐dibenzoate 20‐homovanillylamide. Structurally, RTX‐amide resembles capsaicin more closely than does RTX, and after cleavage of the amide bond the resulting amine would be predicted to not bind to protein kinase C in contrast to resiniferonol 9,13,14‐orthophenylacetate, the parent diterpene of RTX. In contrast to our expectations the binding potency of the RTX‐amide for the vanilloid receptor present in rat spinal cord was 450‐fold lower than that of RTX (Ki values for the RTX‐amide and RTX were 10.4 ± 0.7 nM and 23.1 ± 3.2 pM, respectively). In the case of phorbol 12,13‐dibenzoate 20‐homovanillylamide, there was a further loss of affinity for the vanilloid receptor compared with RTX; nonetheless, the Ki (8.56 ± 0.61 µM) was comparable with that of capsaicin (5.31 ± 0.37 µM). Computer fitting of the binding data yielded Hill coefficient values of 2.25 ± 0.03, 2.33 ± 0.03, and 1.84 ± 0.05 for RTX, RTX‐amide, and phorbol 12,13‐dibenzoate 20‐homovanillylamide, respectively, indicating that both new compounds induced apparent positive cooperativity among vanilloid binding sites. We found that the RTX‐amide was also 20‐ and 300‐fold less potent than RTX in inducing chemogenic pain and hypothermia, respectively. The affinities of the compounds for protein kinase C‐α were evaluated by competition of [3H]phorbol 12,13‐dibutyrate binding. Replacement of the C‐20 hydroxyl group in phorbol 12,13‐dibenzoate by an amine led to a 750‐fold drop in binding affinity, and the conversion of the phorbol 12,13‐dibenzoate 20‐amine to the amide resulted in a further 60‐fold drop in binding activity (Ki values for phorbol 12,13‐dibenzoate, phorbol 12,13‐dibenzoate 20‐amine, and phorbol 12,13‐dibenzoate 20‐homovanillylamide were 0.96 ± 0.01, 720 ± 80, and 43,500 ± 4,060 nM, respectively). The Ki of the RTX‐amide for protein kinase C‐α was >100 µM, whereas RTX and the parent alcohol resiniferonol 9,13,14‐orthophenylacetate yielded Ki values of 1.49 ± 0.19 µM and 36.5 ± 1.3 nM, respectively.
Characterization of Pharmacologically Active Anti‐Peptide Antibodies Directed Against the First and Second Extracellular Loops of the Serotonin 5‐HT1A Receptor
Authors: Verdot L. , Bertin B. , Guilloteau D. , Strosberg A. D. , Hoebeke J.
Published: July 2010

Abstract: The immunological properties and the functional role of the first (loop I) and second (loop II) extracellular loops of the human serotonin 5‐HT1A receptor were studied with three populations of anti‐peptide antibodies: Ab‐1 (loop I; sequence Y‐Q‐V‐L‐N‐K‐W‐T‐L‐G‐Q‐V‐T‐C‐D‐L; residues 96–111), Ab‐2 (loop II; sequence G‐W‐R‐T‐P‐E‐D‐R‐S‐D‐P‐D‐A‐C‐T‐I‐S‐K‐D‐H‐G; residues 173–193), and Ab‐12 (produced against loop I but cross‐reacting with loop II). Chemical modification of peptide amino acid residues revealed the importance of the polyanionic stretch near the N‐terminal domain of loop II for Ab‐2 antibody binding and the role of the cysteine residues in both loops for the binding of Ab‐1 and Ab‐12 antibodies. Antibodies Ab‐2 and Ab‐12 recognized only the nonglycosylated form of the receptor (42 kDa) on immunoblots with transfected HeLa cells expressing the human 5‐HT1A receptor but recognized the glycosylated forms (55 and 65 kDa) of rat 5‐HT1A receptor from hippocampus membranes. The Ab‐1 antibodies recognized no protein band from any cell type studied. Preincubation of transfected HeLa cell membranes with Ab‐2 antibodies revealed two affinity binding sites of the 5‐HT1A receptor (KDH = 0.54 ± 0.09 nM and KDL = 13.74 ± 4.9 nM) for the agonist 8‐hydroxy‐2‐(di‐n‐[3H]propylamino)tetralin ([3H]8‐OH‐DPAT) binding, but Ab‐1 and Ab‐12 revealed only one site (KD of ≈2.5 nM). In contrast to the Ab‐2 antibodies, Ab‐1 and Ab‐12 antibodies decreased the Bmax of the [3H]8‐OH‐DPAT binding to 42 and 31%, respectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high‐affinity state for agonist binding and the other interfering with the accessibility of the ligand binding pocket.
GM1 Ganglioside Activates the High‐Affinity Nerve Growth Factor Receptor trkA
Authors: Rabin Stuart J. , Mocchetti Italo
Published: July 2010

Abstract: The monosialoganglioside GM1 has been shown to possess neurotrophic activity in vitro and in vivo and is now used as an experimental treatment for a variety of neurological disorders and trauma. Little is known about the mechanism of action used by GM1. Because GM1 appears to enhance nerve growth factor (NGF) activity, we have used C6trk+ cells, a derivative of C6‐2B glioma cells that express the high‐affinity receptor for NGF trkA, to determine whether the neurotrophic effects of GM1 occurs through induction of trkA activity. Exposure of C6trk+ cells to NGF (10–50 ng/ml) resulted in a five‐ to 10‐fold increase in trkA tyrosine phosphorylation within 5 min. Incubation of cells with GM1 resulted in a threefold increase in trkA phosphorylation beginning within 1 h and peaking between 3 and 6 h. Optimal responses to GM1 were obtained using 80–100 µM concentrations. Moreover, tyrosine phosphorylation of known trkA target proteins, such as extracellular signal‐regulated kinases, and suc‐associated neurotrophic factor‐induced tyrosine‐phosphorylated target, were activated upon stimulation of C6trk+ cells with GM1. In addition, GM1 potentiated the NGF‐mediated activation of tyrosine phosphorylation of trkA. GM1 failed to induce phosphorylation of trkA and target proteins in mock transfected cells. Thus, our data demonstrate that GM1 mimics some of the effects of NGF and suggest that the neurotrophic properties of GM1 may be attributed to its activation of trkA signal transduction.
Characterization of Nervana, a Drosophila melanogaster Neuron‐Specific Glycoprotein Antigen Recognized by Anti‐Horseradish Peroxidase Antibodies
Authors: Sun Banghua , Salvaterra Paul M.
Published: July 2010

Abstract: Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti‐HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti‐HRP‐reactive Mr 42K glycoprotein that is likely to be the major contributor to neuronal specific anti‐HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti‐HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti‐HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila.
Differential In Vivo Regulation of mRNA Encoding the Norepinephrine Transporter and Tyrosine Hydroxylase in Rat Adrenal Medulla and Locus Ceruleus
Authors: Cubells Joseph F. , Kim Kwang Soo , Baker Harriet , Volpe Bruce T. , Chung Young‐in , Houpt Thomas A. , Wessel Thomas C. , Joh Tong H.
Published: July 2010

Abstract: To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5‐kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine‐secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady‐state levels of norepinephrine transporter mRNA in the adrenal medulla by ∼65% and in the locus ceruleus by ∼25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine depletion.
Transcription Factor AP‐2 Regulates Expression of the Dopamine β‐Hydroxylase Gene
Authors: Greco Dominique , Zellmer Eustacia , Zhang Zheng , Lewis Elaine
Published: July 2010

Abstract: Expression of the gene encoding the neurotransmitter biosynthetic enzyme dopamine β‐hydroxylase (DBH) is regulated in a tissue‐specific pattern, and transcription is influenced by environmental stimuli. Using the promoter proximal region of the rat DBH gene and nuclear extracts from SHSY‐5Y neuroblastoma cells, a DNA‐protein complex was identified that is competitive with oligonucleotides containing the recognition site of transcription factor AP‐2. DNase footprint analysis identified an AP‐2 binding site between −136 and −115 of the DBH promoter. Mutation of that AP‐2 site results in a sevenfold reduction of basal reporter gene expression, but second messenger‐stimulated activity is retained. Cotransfection of an AP‐2 expression vector and a DBH promoter‐reporter construct into cultured cells results in a sixfold stimulation of reporter gene expression, demonstrating the ability of AP‐2 to trans‐activate the DBH promoter. These results identify a new regulatory element on the rat DBH gene and suggest that the AP‐2 site plays a role in maintaining basal levels of DBH transcription.
Enhancement of Neurite Outgrowth Following Calpain Inhibition Is Mediated by Protein Kinase C
Authors: Shea Thomas B. , Cressman Corinne M. , Spencer Melissa J. , Beermann Mary Lou , Nixon Ralph A.
Published: July 2010

Abstract: We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH‐SY‐5Y human neuroblastoma cells. SH‐SY‐5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum‐containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell‐permeant cysteine protease inhibitors N‐acetyl‐leucyl‐leucyl‐norleucinal (“C1”) and calpeptin or by the phospholipid‐mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti‐calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule‐associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2‐O‐tetradecanoylphorbol 13‐acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down‐regulation following TPA treatment. Cell‐free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium‐requiring calpain to cleave the SH‐SY‐5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.