bluuebunny/crossref_metadata_2025 huggingface dataset:
Changing Fatty Acid Content of Growth Cone Lipids Prior to Synaptogenesis
Authors: Martin Rex E. , Bazan Nicolas G.
Published: October 2006

The developing mouse was used to assess biochemical changes in membrane lipids during the period when nerve growth cones become synapses. Growth cone particles and synaptosomes were simultaneously obtained from common brain homogenates. Incorporation of the essential fatty acid, docosahexaenoic acid (22:6ω‐3), was correlated with the developmental changes in endogenous fatty acid content of growth cones and synaptosomes. Analysis of endogenous lipid content indicated that, at all ages studied, the growth cones contained more arachidonoyl acyl chains (20:4ω‐6) than did synaptosomes. Before the onset of synaptogenesis, levels of arachidonoyl chains increased and levels of 22:6, oleoyl and linoleoyl chains decreased in synaptosomes. Although stearoyl and palmitoyl (16:0) remained stable in synaptosomes, 16:0 decreased in growth cones. With the exception of 16:0 and 20:4, endogenous fatty acyl content of growth cones and synaptosomes became similar by postnatal day 10, which coincides with the onset of synaptogenesis. When 5‐day‐old mouse pups were injected intraperitoneally with [3H]22:6, the incorporation into growth cone and synaptosome phospholipids was greatest in phosphatidylethanolamine, followed by phosphatidylser‐ine and phosphatidylcholine. Nominal labeling was present in phosphatidic acid and phosphatidylinositol. Labeling in neutral lipids was less than that of phospholipids, with tria‐cylglycerol incorporating most of the neutral lipid label, followed by diacylglycerol and free 22:6. Only the growth cone fraction contained detectable amounts of 22:6‐labeled cholesterol esters. The distribution of 22:6 label in plasma 72 h after injection indicated that ∼60% of the label was in phospholipids with ∼40% in neutral lipids and <5% in free fatty acids. Collectively, the data indicate that changes in fatty acid composition occur during the conversion of growth cones to synapses.
Enhancement by Cytidine of Membrane Phospholipid Synthesis
Authors: G.‐Coviella Ignacio López , Wurtman Richard J.
Published: October 2006

Cytidine, as cytidine 5′‐diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidyleth‐anolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis‐Menten kinetics (Km= 5 μM; Vmax= 12 × 10−3 mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 μM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse‐chase experiments, addition of cytidine to the medium caused a time‐ and dose‐dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]‐phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p > 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phospha‐tidylethanolamine, and phosphatidylserine, all increasing by 10–15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.
Dopamine Depletion Preferentially Impairs D1 over D2‐Receptor Regulation of Striatal In Vivo Acetylcholine Release
Authors: Bertorelli Rosalia , Zambelli Matilde , Chiara Gaetano Di , Consolo Silvana
Published: October 2006

The roles of D2 and D1 dopaminergic receptors on the regulation of striatal acetylcholine (ACh) release in vivo were examined for a period of 120 min after acute (2 h) or prolonged (16 h) depletion of brain dopamine (DA) by a‐methyl‐p‐tyrosine. The reduction of DA transmission did not affect basal ACh output after 2 h but markedly lowered ACh release by 16 h (50%). Acute α‐methyl‐p‐tyrosine pre‐treatment prevented the reduction of ACh release by the D, antagonist SCH 23390 and its increase by the D2 antagonist, remoxipride, consistent with a drastic reduction of DA transmission at both DA receptors. However, 16 h after α‐methyl‐p‐tyrosine, the effect of remoxipride on ACh release was restored, but SCH 23390 still had no effect, suggesting that the D2 inhibitory tone on ACh release had recovered, whereas the reduction of the D1 facilitatory influence persisted. The D1 facilitatory control of ACh neurotransmis‐sion thus appears to be more sensitive than the D2 inhibitory control to a reduction in DA transmission. The new model of DA‐ACh interaction resulting from these data casts fresh light on the relationship between changes in DA transmission and extrapyramidal motor function.
Glutamate Release in Experimental Ischaemia of the Retina: An Approach Using Microdialysis
Authors: Louzada‐Junior P. , Dias J. J. , Santos W. F. , Lachat J. J. , Bradford H. F. , Coutinho‐Netto J.
Published: October 2006

A rabbit eye model of neural ischaemia is described that uses an increased pressure in the anterior eye chamber to block the capillary supply to the retina. A microdialysis probe placed very close to the retinal surface was used to monitor release of amino acids during ischaemia. A large (two‐ to threefold) increase in the release of glutamate and O‐phosphoserine (twofold), but not of six other amino acids monitored, occurred during initial ischaemia. During reperfusion after release of intraocular pressure, much larger (five‐ to 10‐fold) increases in the release of these amino acids were observed. Parallel ischaemic retinal tissue damage was observed. This damage was prevented by keta‐mine applied locally via a superfusion needle, suggesting that glutamate released during ischaemia, and particularly during reperfusion, was responsible for cell death.
Protein Phosphatases 1 and 2A Dephosphorylate B‐50 in Presynaptic Plasma Membranes from Rat Brain
Authors: Han Yi‐fan , Wang Wei , Schlender Keith K. , Ganjeizadeh Mehdi , Dokas Linda A.
Published: October 2006

The protein B‐50 is dephosphorylated in rat cortical synaptic plasma membranes (SPM) by protein phospha‐tase type 1 and 2A (PP‐1 and PP‐2A)‐like activities. The present studies further demonstrate that B‐50 is dephosphorylated not only by a spontaneously active PP‐1‐like enzyme, but also by a latent form after pretreatment of SPM with 0.2 mM cobalt/20 μg of trypsin/ml. The activity revealed by cobalt/trypsin was inhibited by inhibitor‐2 and by high concentrations (μM) of okadaic acid, identifying it as a latent form of PP‐1. In the presence of inhibitor‐2 to block PP‐1, histone H1 (16–64 μg/ml) and spermine (2 mM) increased B‐50 dephosphorylation. This sensitivity to poly‐cations and the reversal of their effects on B‐50 dephosphorylation by 2 nM okadaic acid are indicative of PP‐2A‐like activity. PP‐1‐ and PP‐2A‐like activities from SPM were further displayed by using exogenous phosphorylase a and histone H1 as substrates. Both PP‐1 and PP‐2A in rat SPM were immunologically identified with monospecific antibodies against the C‐termini of catalytic subunits of rabbit skeletal muscle PP‐1 and PP‐2A. Okadaic acid‐induced alteration of B‐50 phosphorylation, consistent with inhibition of protein phosphatase activity, was demonstrated in rat cortical synaptosomes after immunoprecipitation with affinity‐purified anti‐B‐50 immunoglobulin G. These results provide further evidence that SPM‐bound PP‐1 and PP‐2A‐like enzymes that share considerable similarities with their cytosolic counterparts may act as physiologically important phosphatases for B‐50.
Participation of Prostaglandin E2 in Dopamine D2 Receptor‐Dependent Potentiation of Arachidonic Acid Release
Authors: Di Marzo Vincenzo , Piomelli Daniele
Published: October 2006

Stimulation of dopamine D2 receptors potentiates Ca2+ ionophore‐ or ATP‐induced arachidonic acid (AA) release in D2 receptor cDNA‐transfected Chinese hamster ovary (CHO) cells [CHO(D2)]. By using a combination of chromatographic, biochemical, and radioimmunochemical techniques, we show here that prostaglandin (PG) E2 is a major product of AA metabolism in CHO(D2) cells stimulated with the Ca2+ ionophore A23187. Formation of this PG was markedly increased by the concomitant application of quinpirole, a D2 receptor agonist. In addition, PGE2 enhanced D2‐dependent amplification of AA release, either when it was added (EC50= 100 nM) or when it was produced endoge‐nously, as shown by experiments carried out with the cyclooxygen‐ase inhibitor indomethacin. The results suggest that PGE2 may participate in D2 receptor‐mediated potentiation of AA release in CHO(D2) cells. They also support a functional role for this PG in the modulation of dopaminergic transmission in areas of the CNS, such as amygdala and hypothalamus, where high levels of both PGE2 and dopamine D2 receptors are found.
The Cerebral Microvessels in Culture, an Update
Authors: Joó Ferenc
Published: October 2006

Abstract: Recent advances in our knowledge of the blood–brain barrier (BBB) have in part been made by studying the properties and function of cerebral endothelial cells in vitro. After an era of working with a fraction, enriched in cerebral microvessels by centrifugation, the next generation of in vitro BBB model systems was introduced, when the conditions for routinely culturing the endothelial cells were established. This review summarizes the results obtained from this rapidly growing field. It can be stated with certainty that, in addition to providing a better insight into the chemical composition of cerebral endothelial cells, much has been learned from these studies about the characteristics of transport processes and cell‐to‐cell interactions during the last 12 years. With the application of new technologies, the approach offers a new means of investigation, applicable not only to biochemistry and physiology but also to the drug research, and may improve the transport of substances through the BBB. The in vitro approach has been and should remain an excellent model of the BBB to help unravel the complex molecular interactions underlying and regulating the permeability of the cerebral endothelium.
Characterization of [3H]Ro 5‐4864 Binding Sites in Rat Vas Deferens
Authors: Escubedo E. , Camins A. , Talaveron C. , Camarasa J.
Published: October 2006

Abstract: The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5‐4864 as a radioligand. The binding of [3H]Ro 5‐4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 ± 0.7 × 107M−1 min−1, and the dissociation rate constant (k‐1) was 0.031 ± 0.003 min−1. The dissociation constant (KD) determined by saturation binding was 5.22 ± 0.56 nM. The density of binding was 4,926 ± 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 ± 0.01, an indication that [3H]Ro 5‐4864 binds to a single site. The [3H]Ro 5‐4864 binding was inhibited competitively by Ro 5‐4864 and 2‐hydroxy‐5‐nitrobenzyl‐6‐thioguanosine and noncompetitively by PK 11195, nitrendipine, α,β‐methylene‐ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5‐4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5‐4864 binding sites in rat vas deferens is suggested.
Vestibular Compensation Affects Endogenous Phosphorylation of Frog Brain Proteins
Authors: Janssen U. , Richter‐Landsberg C. , Flohr H.
Published: October 2006

Abstract: The effect of unilateral labyrinthectomy followed by the process of vestibular compensation on the incorporation of radioactive phosphate into frog brain proteins was investigated. Phosphoproteins were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by autoradiography. The present data show that unilateral labyrinthectomy affects the incorporation of 32P into various frog brain proteins. In particular, the phosphorylation of a 20‐kDa protein appeared enhanced during early stages of vestibular compensation (4–12 days). This 20‐kDa protein was shown to be immunologically related to myelin basic protein and its phosphorylation was regulated by an endogenous calcium/calmodulin‐dependent protein kinase. These data might indicate that in addition to neuronal components, components of glial origin are also involved in biochemical events that lead to functional recovery after neuronal lesions.
Increased Cerebrospinal Fluid Dopamine and 3,4‐Dihydroxyphenylacetic Acid Levels in Huntington's Disease: Evidence for an Overactive Dopaminergic Brain Transmission
Authors: Garrett M. C. , Soares‐da‐Silva P.
Published: October 2006

Abstract: Levels of dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), 3‐methoxy‐4‐hydroxyphenylglycol (MHPG), and 5‐hydroxyindoleacetic acid (5‐HIAA) in the CSF of patients with Huntington's disease (HD) were measured by HPLC. CSF DA, DOPAC, and MHPG levels were found to be increased in HD patients. Levels of HVA, 5‐HIAA, and NA in the CSF of HD patients did not differ from those of controls. Changes in CSF DA and DOPAC levels were consistent with previous findings of increased DA tissue content in some brain areas of patients with HD. These results suggest that CSF DOPAC levels could be a more reliable index of over‐active dopaminergic brain systems in HD than CSF HVA levels.
Physiological Levels of Ammonia Regulate Glutamine Synthesis from Extracellular Glutamate in Astrocyte Cultures
Authors: Waniewski Robert A.
Published: October 2006

Abstract: The effect of ammonia on glutamate accumulation and metabolism was examined in astrocyte cultures prepared from neonatal rat cortices. Intact astrocytes were incubated with 70 μMl‐[14C(U)]glutamate and varying amounts of ammonium chloride. The media and cells were analyzed separately by HPLC for amino acids and labelled metabolites. Extracellular glutamate was reduced to 8 μM by 60 min. Removal of glutamate from the extracellular space was not altered by addition of ammonia. The rate of glutamine synthesis was increased from 3.6 to 9.3 nmol/mg of protein/min by addition of 100 μM ammonia, and intracellular glutamate was reduced from 262 to 86 nmol/mg of protein after 30 min. The metabolism of accumulated glutamate was matched nearly perfectly by the synthesis of glutamine, and both processes were proportional to the amount of added ammonia. The transamination and deamination products of glutamate were minor metabolites that either decreased or remained unchanged with increasing ammonia. Thus, ammonia addition stimulates the conversion of glutamate to glutamine in intact astrocyte cultures. At physiological concentrations of ammonia, glutamine synthesis appears to be limited by the rate of glutamate accumulation and the activity of competing reactions and not by the activity of glutamine synthetase.
Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells
Authors: Mutoh Tatsuro , Rudkin Brian B. , Guroff Gordon
Published: October 2006

Abstract: Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor‐sensitive S6 kinase and of the epidermal growth factor‐sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP‐dependent kinase, kinase C, or the calcium/calmodulin‐dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor‐treated cells were also seen on phosphorylation of S6 by cyclic AMP‐dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP‐dependent kinase in vitro, the action of the nerve growth factor‐sensitive S6 kinase was increased about twofold.
In Vivo Distribution of CGS‐19755 Within Brain in a Model of Focal Cerebral Ischemia
Authors: Hogan Matthew J. , Gjedde Albert , Hakim Antoine M.
Published: October 2006

Abstract: The blood–brain barrier permeability of the competitive N‐methyl‐D‐aspartate receptor antagonist CGS‐19755 [cis‐4‐(phosphonomethyl)‐2‐piperidine carboxylic acid] was assessed in normal and ischemic rat brain. The brain uptake index of CGS‐19755 relative to iodoantipyrine was assessed using the Oldendorf technique in normal brain. The average brain uptake index in brain regions supplied by the middle cerebral artery was 0.15 ± 0.35% (mean ± SEM). The unidirectional clearance of CGS‐19755 from plasma across the blood–brain barrier was determined from measurements of the volume of distribution of CGS‐19755 in brain. These studies were performed in normal rats and in rats with focal cerebral ischemia produced by combined occlusion of the proximal middle cerebral artery and ipsilateral common carotid artery. In normal rats the regional plasma clearance across the blood–brain barrier was low, averaging 0.015 ml 100 g−1 min−1. In ischemic rats this clearance value averaged 0.019 ml 100 g−1 min−1 in the ischemic hemisphere and 0.009 ml 100 g−1 min−1 in the nonischemic hemisphere. No significant regional differences in plasma clearance of CGS‐19755 were observed in either normal or ischemic rats except in cortex injured by electrocautery where a 14‐fold increase in clearance across the blood–brain barrier was measured. We conclude that CGS‐19755 crosses the blood–brain barrier very slowly, even in acutely ischemic tissue.
Extracellular Concentration and In Vivo Recovery of Dopamine in the Nucleus Accumbens Using Microdialysis
Authors: Parsons L. H. , Justice J. B.
Published: October 2006

Abstract: The present study compared two different in vivo microdialysis methods which estimate the extracellular concentration of analytes at a steady state where there is no effect of probe sampling efficiency. Each method was used to estimate the basal extracellular concentration of dopamine (DA) in the nucleus accumbens of the rat. In the first method, DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 2.5, 5, and 10 nM) and DA is measured in the dialysate from the brain to generate a series of points which are interpolated to determine the concentration of no net flux. Using this method, basal DA was estimated to be 4.2 ± 0.2 nM (mean ± SEM, n = 5). The slope of the regression gives the in vivo recovery of DA, which was 65 ± 5%. This method was also used to estimate a basal extracellular 3,4‐dihydroxyphenylacetic acid (DOPAC) concentration in the nucleus accumberis of 5.7 ± 0.6 μM, with an in vivo recovery of 52 ± 11% (n = 5). A further experiment which extended the perfusate concentration range showed that the in vivo recovery of DA is significantly higher than the in vivo recovery of DOPAC (p < 0.001), whereas the in vitro recoveries of DA and DOPAC are not significantly different from each other. The in vivo difference is thought to be caused by active processes associated with the DA nerve terminal, principally release and uptake of DA, which may alter the concentration gradient in the tissue surrounding the probe. The second method measures dialysate DA at several perfusion flow rates (0.1, 0.2, 0.4, and 1.2 μ1/min) and extrapolates the data to zero flow using a nonlinear least squares regression. This method estimated a basal extracellular DA concentration of 3.9 ± 0.2 nM (n = 5). The two independent methods are in reasonable agreement that the extracellular concentration of DA in the nucleus accumbens is about 4 nM.
Comparative Metabolism of Fluorinated 3,4‐Dihydroxyphenylalanine Isomers in Aggregating Brain Cell Cultures
Authors: Wiese Claudia , Cogoli‐Greuter Marianne , Weinreich Regin , Winterhalter Kaspar H.
Published: October 2006

Abstract: Fluorinated analogues of 3,4‐dihydroxyphenylalanine (DOPA) were tested for intracellular metabolic conversion in aggregating cell cultures prepared from fetal rat brain. 5‐Fluoro‐D/L‐DOPA was methylated almost exclusively to 3‐O‐methyl‐5‐fluoro‐D/L‐DOPA. Metabolism of 6‐fluoro‐D/L‐DOPA resulted in 6‐fluorodopamine, 6‐fluoro‐3,4‐dihydroxyphenylacetic acid, and 3‐O‐methyl‐6‐fluoro‐D/L‐DOPA, but with a qualitatively and quantitatively different metabolite pattern compared with that of l‐DOPA and D/L‐DOPA, respectively. Homovanillic acid and fluorohomovanillic acid have not been found intracellularly in the cultures. On the basis of these data, the model development of the cerebral metabolism of tracers used in positron emission tomography can be improved.
Characterization of Radioiodinated TISCH: A High‐Affinity and Selective Ligand for Mapping CNS D1 Dopamine Receptor
Authors: Billings Jeffrey J. , Kung Mei‐Ping , Chumpradit Sumalee , Mozley David , Alavi Abass , Kung Hank F.
Published: October 2006

Abstract: In developing CNS D1 dopamine receptor‐imaging agents with improved specificity and longer brain retention, an iodinated D1 ligand was synthesized. In vitro and in vivo radiolabeling studies of a new iodinated benzazepine, TISCH [7‐chloro‐8‐hydroxy‐1‐(3′‐iodophenyl)‐3‐methyl‐2,3,4,5‐tetrahydro‐1 H‐3‐benzazepine], an analog of SCH 23390 (7‐chloro‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine), were investigated. After an intravenous injection, the R(+) isomer of TISCH showed high brain uptake in rats (2.20 and 0.57% dose per whole brain at 2 and 60 min, respectively). The striatum/cerebellum ratio increased progressively with time (12 at 60 min). Ex vivo autoradiography of rat brain sections, after intravenous injection of R(+)‐[125I]TISCH, displayed the highest uptake in striatum and substantia nigra, regions known to have a high concentration of D1 receptors, whereas the S(–) isomer displayed no specific uptake. Furthermore, the specific uptake can be blocked by pretreatment with SCH 23390. In vitro binding studies using the rat striatum tissue preparation showed high specific and low nonspecific bindings (KD= 0.21 ± 0.03 nM). The rank order of potency exhibiting high specificity to the D1 receptor was SCH 23390 > (±)‐TISCH > (+)‐butaclamol = (±)‐FISCH [7‐chloro‐8‐hydroxy‐1‐(4′‐iodophenyl)‐3‐methyl‐2,3,4,5‐tetrahydro‐1 H‐3‐benzazepine] ≥ WB4101 = spiperone > dopamine, serotonin, (±)‐propranolol, and naloxone. Imaging studies in a monkey with the resolved isomer, R(+)‐[123I]TISCH, demonstrated a high uptake in the basal ganglia and prolonged retention. The preliminary data suggest that R(+)‐TISCH is selective for the CNS D1 receptor and is potentially useful for in vivo and in vitro pharmacological studies. When labeled with iodine‐123, it may be suitable for noninvasive imaging in humans.
[3H]Paroxetine Binding and Serotonin Content of Rat Cortical Areas, Hippocampus, Neostriatum, Ventral Mesencephalic Tegmentum, and Midbrain Raphe Nuclei Region Following p‐Chlorophenylalanine and p‐Chloroamphetamine Treatment
Authors: Dewar Karen M. , Grondin Louise , Carli Martin , Lima Lucimey , Reader Tomás A.
Published: October 2006

Abstract: The agents p‐chlorophenylalanine (PCPA) and p‐chloroamphetamine (PCA) deplete brain serotonin (5‐HT) levels by two different mechanisms; PCPA inhibits the enzyme tryptophan hydroxylase, whereas PCA has a neurotoxic action on certain 5‐HT neurons. The parameters of [3H]paroxetine binding to homogenates prepared from the cerebral cortex of rats treated with PCPA, PCA, or saline vehicle were investigated. The tissue concentrations of 5‐HT and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) were also determined by HPLC in the same brain samples. After PCPA treatment, neither the maximum binding capacity (Bmax) nor the dissociation constant (KD) of [3H]paroxetine for the 5‐HT uptake recognition site differed from controls despite a substantial reduction in the concentration of 5‐HT and 5‐HIAA. In contrast, significant changes in both the Bmax and KD values were observed in the cerebral cortex of rats treated with PCA. Furthermore, [3H]paroxetine binding and tissue concentrations of 5‐HT and 5‐HIAA were measured in the following different regions of the rat brain: cingulate, parietal, and visual cortical areas; dorsal and ventral hippocampus; rostral and caudal halves of neostriatum; ventral mesencephalic tegmentum; and midbrain raphe nuclei region after administration of PCPA, PCA, or saline vehicle. There was an excellent correlation between regional 5‐HT levels and specific [3H]paroxetine binding in control and PCA‐treated rats although this correlation was lost after PCPA treatment. Under these conditions, the 5‐HT innervation remains unchanged whereas the concentration of 5‐HT and 5‐HIAA is greatly reduced. Thus, [3H]paroxetine binding appears to provide a reliable marker of 5‐HT innervation density within the mammalian CNS.
Carbon Dioxide Fixation in Neuronal and Astroglial Cells in Culture
Authors: Kaufman Elaine E. , Driscoll Bernard F.
Published: October 2006

Abstract: The concentration of potassium in the extracellular fluid has been found to stimulate the rate of CO2 fixation by astroglial cells grown in primary culture. Raising the concentration of extracellular potassium increased both the initial rate of formation of the 14C‐labeled products of 14CO2 fixation and the final steady‐state level of these products within the cells. In contrast, neither veratridine nor l‐glutamate affected the rate of CO2 fixation in astroglial cells. The very low rate of CO2 fixation found in primarily neuronal cultures was unaffected by increased extracellular potassium as was CO2 fixation in fibroblasts. When cultured alone, astroglial cells release a large fraction of the 14C‐labeled products of CO2 fixation into the surrounding medium. Mixed cultures of astroglia and neurons also fix CO2 but, in contrast to astroglia cultured alone, release only a small fraction of the 14C‐labeled products into the culture medium.
Transmitter‐Like Basal and K+‐Evoked Release of 3,4‐Dihydroxyphenylalanine from the Striatum in Conscious Rats Studied by Microdialysis
Authors: Nakamura S. , Goshima Y. , Yue J. L. , Misu Y.
Published: October 2006

Abstract: Using microdialysis and HPLC, characteristics of the release of endogenous 3,4‐dihydroxyphenylalanine (DOPA) from striatum in conscious rats were studied in comparison with those of 3,4‐dihydroxyphenylethylamine (dopamine; DA). Purified l‐aromatic amino acid decarboxylase (AADC) converted a putative peak of DOPA to DA. The retention time of DOPA differed from that of DA and major metabolites of DA and norepinephrine. The DOPA peak of dialysates comigrated with that of authentic DOPA when the pH of the HPLC buffer was modified. The ratio of the basal release of DOPA:DA was 1:2. 3‐Hydroxybenzyl‐hydrazine (NSD‐1015; 100 mg/kg, i.p.), an AADC inhibitor, markedly increased the basal release of DOPA but produced no effect on DA. The basal release of DOPA was markedly decreased by α‐methyl‐p‐tyrosine (200 mg/kg, i.p.), substantially tetrodotoxin (1 μM) sensitive, and Ca2+ (removal plus 12.5 mM Mg2+ addition) dependent. Fifty millimolar K+ released DOPA and this release was also Ca2+ dependent. These characteristics of the basal and evoked release of DOPA were similar to those of DA. The ratio of the evoked release of DOPA:DA was 1:3. These results indicate that DOPA is released under physiological conditions and by K+‐induced depolarization in a manner similar to that for transmitter DA from striatum in freely moving rats.
Enhanced Tyrosine Hydroxylation in Hippocampus of Chronically Stressed Rats upon Exposure to a Novel Stressor
Authors: Nisenbaum Laura K. , Abercrombie Elizabeth D.
Published: October 2006

Abstract: We have used microdialysis to measure the in vivo level of tyrosine hydroxylation in hippocampus of the freely moving rat. An inhibitor of aromatic amino acid decarboxylase, NSD‐1015, was administered through the dialysis probe and the resulting accumulation of 3,4‐dihydroxyphenylalanine (DOPA) in extracellular fluid of hippocampus was quantified. Administration of the tyrosine hydroxylase inhibitor, α‐methyl‐p‐tyrosine, decreased extracellular DOPA to undetectable levels. In addition, both systemic and local application of clonidine, an α2‐adrenergic agonist, produced a decrease in extracellular DOPA. In response to acute tail shock, a significant increase in extracellular DOPA was observed. Thus, it appears that in vivo accumulation of DOPA after local administration of NSD‐1015 provides a reliable index of hippocampal tyrosine hydroxylation. We have used this technique to investigate whether prior exposure to chronic stress alters the in vivo level of tyrosine hydroxylation in hippocampus under basal conditions as well as in response to a novel stressor. In rats previously exposed to chronic cold stress, the basal accumulation of extracellular DOPA did not differ from naive controls. Acute tail shock, however, produced a significantly greater and more prolonged elevation in extracellular DOPA of chronically stressed rats. These data suggest that enhanced biosynthetic capacity of noradrenergic terminals may be one mechanism underlying adaptation to chronic stress.
Inhibition of Ischemia‐Induced Dopamine Release by ω‐Conotoxin, a Calcium Channel Blocker, in the Striatum of Spontaneously Hypertensive Rats: In Vivo Brain Dialysis Study
Authors: Ooboshi Hiroaki , Sadoshima Seizo , Yao Hiroshi , Nakahara Tatsuo , Uchimura Hideyuki , Fujishima Masatoshi
Published: October 2006

Abstract: The effect of ω‐conotoxin GVIA (CgTX), an N‐and l‐type voltage‐sensitive calcium channel (VSCC) blocker, on the release of dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC) in the striatum before and during transient cerebral ischemia in spontaneously hypertensive rats was studied using an in vivo brain dialysis technique. Continuous perfusion of CgTX in the striatum was started 20 min before ischemia and concentrations of dopamine and DOPAC in the dialysate were measured using HPLC with an electrochemical detector. Before ischemia, both 10 and 100 μM CgTX significantly lowered the concentration of dopamine, to 49% of the basal values, DOPAC concentrations also decreased significantly, by 28 and 17%, respectively. Forebrain ischemia, produced by bilateral carotid artery occlusion, reduced striatal blood flow to less than 6% of the resting value in each group. During 20 min of ischemia, the vehicle group showed a marked increase in dopamine (175 times the basal concentration). In the 10 or 100 μM CgTX perfusion group, in contrast, dopamine release was significantly attenuated, to 38 or 29% of the vehicle group, respectively. DOPAC concentrations decreased during ischemia to 58% of the basal value in the vehicle group and 49% in both CgTX groups. These results indicate that the massive release of striatal dopamine during ischemia depends largely on the influx of extracellular calcium via CgTX‐sensitive VSCCs.
21‐Aminosteroids Interact with the Dopamine Transporter to Protect Against 1‐Methyl‐4‐Phenylpyridinium‐Induced Neurotoxicity
Authors: Sanchez‐Ramos Juan R. , Song Shije , Mash Deborah C. , Weiner William J.
Published: October 2006

Abstract: U‐78518F, a 21‐aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin l‐methyl‐4‐phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U‐78518F up to 30 μM. Nonspecific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U‐78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of γ‐[3H]aminobutyric acid. In human striatal membrane preparations, U‐78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 μM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21‐aminosteroids in MPP+‐induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.
Morphometric Analysis of Normal, Mutant, and Transgenic CNS: Correlation of Myelin Basic Protein Expression to Myelinogenesis
Authors: Shine H. David , Readhead Carol , Popko Brian , Hood Leroy , Sidman Richard L.
Published: October 2006

Abstract: The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber. These data suggest that oligodendroglial function is governed in part by the degree of MBP expression.
c‐fos Expression as a Model for Studying the Action of Hexachlorocyclohexane Isomers in the CNS
Authors: Vendrell M. , Tusell J. M. , Serratosa J.
Published: October 2006

Abstract: The induction of protooncogene c‐fos in the CNS after administration of several convulsants has been studied. The organochlorine insecticide γ‐hexachlorocyclohexane (γ‐HCH, lindane) has been shown to induce c‐fos expression in different brain areas. Pentylenetetrazole and picrotoxin, a known γ‐aminobutyric acid‐receptor antagonist, have also been considered. The administration of two nonconvulsant isomers of γ‐HCH, α‐HCH, and δ‐HCH, before the mentioned toxicants, affects the protooncogene expression in different ways. The differential pattern of expression displayed by c‐fos after these treatments suggests the presence of diverse mechanisms of action for the compounds studied.
A Highly Sensitive Assay for Histamine Using Ion‐Pair HPLC Coupled with Postcolumn Fluorescent Derivatization: Its Application to Biological Specimens
Authors: Itoh Yoshinori , Oishi Ryozo , Adachi Naoto , Saeki Kiyomi
Published: October 2006

Abstract: A simple and highly sensitive method for the determination of histamine (HA) was developed using ion‐pair, reversed‐phase HPLC coupled with postcolumn o‐phthalal‐dehyde derivatization fluorometry, and it was applied to the unpurified extracts of human and rat plasma, and brains of rats and mice. The HA concentrations both in the plasma and brains determined by the present method were well consistent with the values obtained by cation‐exchange HPLC with postcolumn fluorescent derivatization currently in use. The present method was more advantageous than the assay using cation‐exchange HPLC: (1) it was three to four times more sensitive (the detection limit was 0.5 pg of HA), and (2) it enabled the measurement of HA in samples containing C/?)a‐methylhistamine, a potent and specific H3‐receptor agonist, which could not be separated from HA by cation‐exchange chromatography. Using the present method coupled with intracerebral microdialysis, we found in the rat hypo‐thalamus that (R)α‐methylhistamine (5 mg/kg i.p.) markedly decreased the extracellular concentration of HA with a maximal effect (83% reduction) during 30‐60 min after injection, suggesting that most of HA in the microdialysate fraction is neuronal in origin.
Brain Transfer Coefficients for 67Ga: Comparison to 55Fe and Effect of Calcium Deficiency
Authors: Murphy Vincent A. , Rapoport Stanley I.
Published: October 2006

Abstract: The transfer coefficients (Kia) for the uptake of gal‐lium‐67 (67Ga) into brain and CSF were determined in un‐anesthetized male Fischer‐344 rats fed either a normal or a low‐Ca diet. Kin for 67Ga was also compared with transfer coefficients for the uptake of iron‐55 (55Fe) and 125I‐albumin in control animals. The value of CSF 67Ga Kin was 3 × 10−7 ml·g−1·s−1 and was 50% larger in low‐Ca animals. Brain regional Kin values for 67Ga were 3‐9 × 10−7 ml·g−1·s−1 with no differences in Kia between normal and low‐Ca rats. CSF Km values for 55Fe were 40% and those for albumin were 15% of Kia for 67Ga. For brain, Kin values for 55Fe were 15‐40% smaller than for 67Ga, but for albumin the Kin values were 85% less than for 67Ga. 67Ga was found to be 99% bound to plasma proteins, whereas 55Fe was 99.9% bound. The results indicate that metals that are primarily bound to trans‐ferrin enter the CSF and brain very slowly. Uptake of both metals was faster than albumin, which may indicate that metal bound to small chelates contributes significantly to brain uptake. In addition, Ca deficiency does not enhance entry of Ga into the brain.
γ‐Glutamyl Transpeptidase Activity in Brain Microvessels Exhibits Regional Heterogeneity
Authors: Wolff Johannes E. A. , Belloni‐Olivi Luisa , Bressler Joseph P. , Goldstein Gary W.
Published: October 2006

Abstract: Brain microvessels form a tight blood‐tissue permeability barrier and express high levels of specific enzymes, including γ‐Glutamyl transpeptidase (GGTP). This differentiation is thought to be induced by perivascular astrocytes. By using histochemical methods, we found that the percentage of GGTP‐positive vessels varied considerably in different areas of rat brain. Enzyme activity was not found in the pineal gland or the median eminence, where the blood‐brain barrier is not expressed. In areas where the blood‐brain barrier is expressed, the percentage of GGTP‐positive vessels varied from 8% in the optic nerve to 100% in the anterior commissure. The neocortex showed a lower percentage of GGTP‐positive vessels (2‐15%) than anterior olfactory nucleus (42%), subiculum (70%), hippocampus (48%), and striatum (50‐58%). Alkaline phosphatase, another brain microvessel‐enriched enzyme, did not show these marked regional differences. The morphometric histochemical results were verified by enzymatic assays in homogenates of different regions from rat and bovine brain and in microvessel preparations of bovine putamen and neocortex. During the postnatal development of rat brain, the difference between neocortex and striatum appeared after day 20. The regional heterogeneity of brain microvessels may be caused by astrocytic heterogeneity and reflect regional heterogeneity in microvascular function.
Opioid Control of the In Vitro Release of Cholecystokinin‐Like Material from the Rat Substantia Nigra
Authors: Benoliel J. J. , Mauborgne A. , Bourgoin S. , Legrand J. C. , Hamon M. , Cesselin F.
Published: October 2006

Abstract: Possible interactions between Met‐enkephalin and cholecystokinin (CCK)‐containing neurons in the rat sub‐stantia nigra were investigated by looking for the effects of various opioid receptor ligands and inhibitors of enkephalin‐degrading enzymes on the K+‐evoked overflow of CCK‐like material (CCKLM) from substantia nigra slices. The δ‐opioid agonists d‐Pen2,dPen5‐enkephalin (50 μM) and Tyr‐D‐Thr‐Gly‐Phe‐Leu‐Thr (DTLET; 3 μM) enhanced, whereas the n‐opioid agonists Tyr‐D‐Ala‐Gly‐MePhe‐Gly‐ol (DAGO; 10 μM) and MePhe3, D‐Pro4‐morphiceptin (PL 017; 10 μM) decreased, the K+‐evoked release of CCKLM. By contrast, the δ‐opioid agonist U‐50488 H (5 μM) was inactive. The stimulatory effect of DTLET could be prevented by the 5 antagonist ICI‐154129 (50 μM but not by the μ antagonist naloxone (1 μM). Conversely, the latter drug, but not ICI‐154129, prevented the inhibitory effect of DAGO and PL 017. A significant increase in CCKLM overflow was observed upon tissue supervision with the peptidase inhibitors kelatorphan or bestatin plus thiorphan. This effect probably resulted from the stimulation of δ‐opioid receptors by endogenous enkephalins protected from degradation, because it could be prevented by ICI‐154129 (50 μM Furthermore, the peptidase inhibitors did not enhance CCKLM release further when S‐opioid receptors were stimulated directly by DTLET (3 μM). These data indicate that opioids acting on d and n receptors may exert an opposite influence, i.e., excitatory and inhibitory, respectively, on CCK‐containing neurons in the rat substantia nigra. Because CCK has anti‐opioid properties, the δ‐opioid control of CCKLM release might participate in the central mechanisms of opiate tolerance.
Tau 2: A Probe for a Ser Conformation in the Amino Terminus of τ
Authors: Watanabe Nobuo , Takio Koji , Hasegawa Masato , Aral Takao , Titani Koiti , Ihara Yasuo
Published: October 2006

Abstract: We have determined the epitope for Tau 2, a monoclonal antibody that intensely stained tangles, plaque neurites, and curly fibers in the tissue section, and strongly labeled bovine τ, but only very weakly labeled human r on the blot. The epitope has been localized to Ala95 through Ala108 of bovine τ. Ser101 is critical for Tau 2 reactivities; the replacement of Ser by Pro, which is found in rat, mouse, and human T, brings about very weak Tau 2 reactivities. The strong Tau 2 staining of tangles and its effective absorption with a synthetic Ser peptide (Ala95 through Ala108) suggest that the τ in paired helical filaments takes a Ser conformation, rather than a Pro conformation, in its ammo‐terminal portion.
Detection of Mobile Proteins by Proton Nuclear Magnetic Resonance Spectroscopy in the Guinea Pig Brain Ex Vivo and Their Partial Purification
Authors: Kauppinen Risto A. , Kokko Hani , Williams Stephen R.
Published: October 2006

Abstract: Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to study metabolites of the brain cortex ex vivo. The superfused brain cortex preparation was judged to be metabolically viable on the basis of the 31P NMR spectrum (intracellular pH of 7.23 ± 0.03 and phosphocreatine/ ATP ratio of 1.21 ± 0.09). Using‘H NMR a group of previously unidentified signals was detectable at 0.94, 1.22, and 1.40 ppm with a water‐suppressed spin‐echo sequence. These signals had shorter spin‐spin relaxation times (51‐54 ms) than TV‐acetylaspartate and lactate (84‐93 ms) and also smaller saturation factors, an indication of shorter spin‐lattice relaxation times than the latter two low‐molecular‐weight metabolites. The unidentified signals also displayed homo‐nuclear coupling to other spins in the methine region of the spectrum. Acid extraction of the brain slices or cortex from animals that were killed yielded a mixture of proteins that exhibited NMR properties matching the 1H NMR signals in the brain cortex. The molecular mass of these thermoresistant, “mobile’ proteins, which contained proline plus hydroxy‐proline (9‐16% of all amino acids), ranged between 8 and 40 kDa. These “new’ assigμMents of1H NMR‐detectable compounds may influence interpretation of NMR data of some metabolites, as their signals are in the vicinity of the ‐CH3 1H NMR peaks of lactate and alanine.
Characterization of Neutral Proteinases from Alzheimer‐Affected and Control Brain Specimens: Identification of Calcium‐Dependent Metalloproteinases from the Hippocampus
Authors: Backstrom Jon R. , Miller Carol A. , Tökés Zoltán A.
Published: October 2006

Abstract: Three neutral proteinases from human hippocam‐pal tissue have been identified and partially characterized using substrate gel electrophoresis. The proteinases showed activity when gelatin was used as the substrate, but had no detectable activity against casein. Based on the results of inhibition studies and the calcium requirements, it was concluded that the activities were due to calcium‐dependent me‐talloproteinases. The apparent molecular weights were 130,000 (MP‐130), 100,000 (MP‐100), and 70,000 (MP‐70). Half‐maximal activities were observed with 20 μM Ca2+ for MP‐130,40 μM Ca2+ for MP‐100, and 800 μM Cn2+ for MP‐70. In the presence of Ca2+, Zn2+ reestablished the activities of the three metalloproteinases at a lower concentration than did either Co2+ or Mn2+. One millimolar Al3+ inhibited 67% of the MP‐70 activity, but did not affect the MP‐100 and MP‐130 activities. An analysis of Alzheimer‐affected hip‐pocampal and control samples showed that the specific activity (in units per milligram of sodium dodecyl sulfate‐soluble protein) of MP‐70 varied less than the activities of MP‐100 and MP‐130 between the two groups. Although p‐amino‐phenylmercuric acetate (p‐APMA) increased the activities of MP‐70 by 70% in both groups of specimens, the resulting activities from Alzheimer samples were greater than those from control samples (p < 0.01). A wide range of MP‐100 specific activity was observed in both groups, and its mean activity was higher in Alzheimer‐affected samples (p < 0.05). Treatment with p‐APMA increased the activity of MP‐100 only 25% in both groups of tissue samples. MP‐130 activity was detected predominantly in Alzheimer‐affected hippo‐campal specimens, and treatment with p‐APMA failed to increase its activity in both the control and the Alzheimer‐affected specimens. The results demonstrate an elevated level of metalloproteinase activities, capable of degrading tissue matrix components, in the hippocampus from postmortem Alzheimer patients.
Steroid Inhibition of Neural Micro vessel Morphogenesis In Vitro: Receptor Mediation and Astroglial Dependence
Authors: Wolff Johannes E. A. , Laterra John , Goldstein Gary W.
Published: October 2006

Abstract: Steroid hormones alter several aspects of micro‐vascular function within the CNS. Both microvessel formation and blood‐brain barrier expression appear to be influenced by interactions between astrocytes and endothelial cells. To determine if steroids alter astrocyte‐endothelial interactions, we studied their effects on astroglial‐induced micro‐vessel morphogenesis in vitro. Q astroglial cells induce bovine retinal microvascular endothelial cells to differentiate into capillary‐like structures. Dexamethasone, hydrocortisone, and progesterone at 10 nM inhibited C6‐induced microvessel morphogenesis by 75, 35, and 30%, respectively. Inhibition by dexamethasone was both time and concentration dependent, reaching 80‐100% at 1 μM. Tetrahydrocortisone and 17α‐hydroxyprogesterone had only marginal inhibitory effects. Cortexolone, a glucocorticoid receptor antagonist, blocked inhibition by dexamethasone. Progesterone receptors were expressed in C6 but not bovine retinal microvascular endothelial cells, identifying the astroglial cell as the likely effector of progesterone‐mediated inhibition. Astroglial cells were further implicated as the effectors of steroid‐mediated inhibition because none of the steroids inhibited astroglial‐independent capillary‐like structure formation in response to a reconstituted extracellular matrix, Matrigel. These findings are evidence that steroids modulate neural microvascular endothelial cell functions indirectly through perivascular astrocytes via a receptor‐mediated mechanism.