bluuebunny/crossref_metadata_2025 huggingface dataset:
[3H]Nipecotic Acid Binding to γ‐Aminobutyric Acid Uptake Sites in Postmortem Human Brain
Authors: Czudek Carole , Reynolds Gavin P.
Published: October 2006

Abstract: Binding of [3H]nipecotic acid, a proposed marker for GABAergic neurons, was investigated in postmortem human brain by use of a centrifugation assay. Binding was displaceable, apparently saturable, and to a single site, with typical KD and Bmax values of 1.85 μM and 124.2 pmol/mg of protein in the hippocampus. Regional distribution studies indicated a heterogeneous population of [3H]nipecotic acid binding sites with highest concentrations in the lateral globus pallidus. Putamen tissue from four cases of Huntington's disease showed a marked reduction in [3H]nipecotic acid binding. Binding correlated with both age and postmortem delay in the hippocampus. There was an effect of agonal state in which prolonged illness before death apparently caused a reduction in binding. Our results indicate that [3H]nipecotic acid may be used successfully as a marker for neuronal GABAergic uptake sites in human brain, but that the effects of variables such as age, postmortem delay, and agonal state must always be taken into account.
Role of Adenylate Cyclase in the Modulation of the Release of Dopamine: A Microdialysis Study in the Striatum of the Rat
Authors: Santiago M. , Westerink B. H. C.
Published: October 2006

Abstract: In the present study, we have applied the brain microdialysis technique to investigate the effect of the stimulation of adenylate cyclase on the extracellular levels of dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA) in the striatum of freely moving rats. Infusion of 8‐bromo‐adenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP), 3‐isobutyl‐1‐methylxanthine, or forskolin produced a significant increase in the release of DA. The effect of 8‐Br‐cAMP was tetrodotoxin, Ca2+, and dose dependent and was saturable. 8‐Br‐cAMP also caused an increase in the output of DOPAC and HVA. No effects were seen on the output of 5‐HIAA, except at the highest 8‐Br‐cAMP concentration studied. Infusion of 8‐Br‐cAMP (25 μM, 1.0 mM, and 3.3 mM) together with infusion of (–)‐sulpiride (1 μM) or systemic administration of (±)‐sulpiride (55 μmol/kg i.p.) produced an additive effect on the release of DA. Infusion or peripheral administration of (–)‐N‐0437 (1 μM or 1 μmol/kg) both decreased the 8‐Br‐cAMP‐induced increase in the release of DA. These results demonstrate that cyclic AMP may stimulate the release of DA, but it is unlikely that this second messenger is linked to presynaptic D2 receptors controlling the release of DA.
Effect of Ammonia on Brain Serotonin Metabolism in Relation to Function in the Portacaval Shunted Rat
Authors: Bergeron Marcelle , Swain Margaret S. , Reader Tomás A. , Grondin Louise , Butterworth Roger F.
Published: October 2006

Abstract: Four weeks following portacaval anastomosis (PCA) in the rat, severe liver atrophy, sustained hyperammonemia, and increased plasma and brain tryptophan are observed. Administration of ammonium acetate (NH4Ac) to rats with PCA precipitates severe signs of hepatic encephalopathy (HE) (loss of righting reflex progressing to loss of consciousness and ultimately deep coma). To evaluate the relationship between the deterioration of neurological status in HE and serotonin (5‐HT) metabolism, the levels of 5‐HT, its precursor 5‐hydroxytryptophan, and its major metabolite 5‐hydroxyindole‐3‐acetic acid (5‐HIAA) were measured by HPLC with ion‐pairing and electrochemical detection in three well‐defined areas of the cerebral cortex: anterior cingulate, piriform and entorhinal, and frontoparietal; as well as in the caudateputamen, the raphe nuclei, and the locus ceruleus in rats with PCA at different stages of HE, before and after injection of NH4Ac, as well as in sham‐operated controls. The results demonstrate increased 5‐HIAA/5‐HT ratios after PCA and NH4Ac loading, suggesting increased 5‐HT turnover in the brains of these animals. However, these changes do not appear to be related to the precipitation of coma as no significant difference in 5‐HT turnover was observed between precoma and coma stages of HE. Increased 5‐HT turnover in brain of shunted rats may be related to early symptoms of HE such as altered sleep patterns and disorders of motor coordination.
Kinetics and Block of Dopamine Uptake in Synaptosomes from Rat Caudate Nucleus
Authors: Krueger Bruce K.
Published: October 2006

Abstract: The dopamine (DA) uptake system in mammalian nerve terminals was studied by measuring the unidirectional influx of tritiated DA into synaptosomes prepared from rat caudate nucleus. Two distinct time‐dependent components of DA uptake were observed. The principal component was saturable with respect to DA concentration, required both external Na and Cl, and was competitively blocked by micromolar concentrations of the psychotropic agents cocaine, benztropine, nomifensine, amphetamine, and methamphetamine. This principal component of uptake has the properties expected for a carrier‐mediated transport system. The second component, which accounted for about 10–30% of the DA uptake at 2 μM DA, was not saturable, and was independent of external Na, Cl, and blockers of the carrier‐mediated system. The saturable, Na‐dependent component had an apparent Km(DA) of about 0.5 μM The dependence of DA uptake on external Na was sigmoid [Hill coefficient = 2; Ka(Na) = 45 mM] whereas the dependence on Cl was best described by a rectangular hyperbola [Ka(Cl) = 15 mM]. Depolarizing conditions (elevated external K) reduced the rate of DA influx. The data are consistent with a carrier‐mediated DA transport mechanism in which each DA molecule entering the nerve terminal via the carrier is accompanied by two or more Na ions and one Cl ion in a rheogenic process carrying one or more net positive charges into the cell. Net, concentrative accumulation of DA inside nerve terminals may be accomplished by utilizing the Na electrochemical gradient to drive DA against its electrochemical gradient via this carrier system.
Inhibition of Nitric Oxide Synthase Blocks N‐Methyl‐D‐Aspartate‐, Quisqualate‐, Kainate‐, Harmaline‐, and Pentylenetetrazole‐Dependent Increases in Cerebellar Cyclic GMP In Vivo
Authors: Wood Paul L. , Emmett Mark R. , Rao Tadimeti S. , Cler Julie , Mick Steve , Iyengar Smriti
Published: October 2006

Abstract: The synthesis of nitric oxide by brain slices has been demonstrated in several laboratories. In addition, in vitro studies have demonstrated stimulation of nitric oxide synthesis by excitatory amino acid receptor agonists. These data have led to the hypothesis that this readily diffusible “intercellular messenger molecule”acts to generate a cascade effect by activating guanylate cyclase in several cell types and thereby augment levels of the second messenger cyclic GMP (cGMP). Therefore, we evaluated this hypothesis in vivo, by testing the actions of the nitric oxide synthase inhibitor N‐monomethyl‐L‐arginine (NMMA) on elevations in level of mouse cerebellar cGMP generated by excitatory amino acid receptor agonists. The stimulatory effects of D‐serine, quisqualate, and kainate were all found to be antagonized by this enzyme inhibitor. In addition, NMMA antagonized the increases in cerebellar cGMP level elicited by harmaline and pentylenetetrazole, pharmacological agents that augment endogenous excitatory amino acid transmission. Our data are, therefore, the first in vivo demonstration that nitric oxide is an important “messenger molecule”in the cerebellum, mediating the actions of kainate, quisqualate, and N‐methyl‐D‐aspartate receptor agonists on guanylate cyclase. These data are consistent with previous in vitro findings with kainate and N‐methyl‐D‐aspartate.
Stereotaxic Injection of Tetanus Toxin in Rat Central Nervous System Causes Alteration in Normal Levels of Monoamines
Authors: Aguilera José , López Lluis Arístides , González‐Sastre Francesc
Published: October 2006

Abstract: A single intraventricular injection of tetanus toxin produced a time‐dependent elevation of serotonin levels in brain and spinal cord of adult rats. This tetanus toxin‐induced increase was produced in areas of high density of serotonergic innervation, such as the hypothalamus, hippocampus, and spinal cord. Little or no effect was found in the thalamus, cerebellum, and frontal cortex, areas that are poorly innervated by serotonergic terminals. The responses of catechol‐amines (no change in dopamine level and generalized decrease in norepinephrine) pointed to a specific action of tetanus toxin on the serotonergic system. Stereotaxic injections of tetanus toxin in dorsal or magnus raphe nuclei did not have an evident effect on biogenic amine levels in the brain and spinal cord, respectively. Because direct stereotaxic injections of the toxin in the hypothalamus or hippocampus produced significant serotonin increases in both areas, it is proposed that tetanus toxin interacts with presynaptic targets to produce serotonin accumulation; this is probably due in part to an activation of tryptophan 5‐hydroxylase.
The Anxiogenic β‐Carboline FG 7142 Selectively Increases Dopamine Release in Rat Prefrontal Cortex as Measured by Microdialysis
Authors: Bradberry Charles W. , Lory John D. , Roth Robert H.
Published: October 2006

Abstract: The effect of the anxiogenic β‐carboline methyl‐β‐carboline‐3‐carboxyamide (FG 7142) on dopamine release in prefrontal cortex and striatum in the awake freely moving rat was determined using the technique of microdialysis. FG 7142 (25 mg/kg, i.p.) caused a time‐dependent increase in dopamine release in prefrontal cortex which was statistically significantly greater than the response to vehicle administration. Dopamine release in striatum was unaltered by FG 7142. Pretreatment of animals with the benzodiazepine antagonist Ro 15‐1788 (30 mg/kg, i.p., 15 min prior to FG 7142 administration) completely abolished the increase in dopamine release caused by FG 7142 in prefrontal cortex. These data indicate that the anxiogenic benzodiazepine inverse agonist FG 7142 can selectively increase dopamine release in prefrontal cortex, and that this effect appears to be mediated via the γ‐aminobutyric acid/benzodiazepine receptor complex.
Identification of Meningeal Cell Released Neurite Promoting Activities for Embryonic Hippocampal Neurons
Authors: Matthiessen H. Peter , Schmalenbach Corinne , Müller Hans Werner
Published: October 2006

Abstract: Primary cultures of meningeal cells from embryonic rat cerebra secrete neurite growth‐inducing components into serum‐free culture medium. This conditioned medium (CM) was analyzed by FPLC and immunochemical and enzymatic treatments and tested for neurite promoting activity (NPA) in a quantitative bioassay using hippocampal neurons from embryonic rat. By immunoprecipitation or specific adsorption we identified laminin (LN)‐proteoglycan complexes and fibronectin (FN), respectively, as the major neurite promoting components within meningeal cell CM. The LN‐proteoglycan complexes and their NPA were sensitive to chondroitinase (chondroitin ABC lyase, EC 4.2.2.4) and to a smaller extent to heparitinase (heparitin sulfate lyase, EC 4.2.2.8). Minor fractions of the total NPA in CM correlated with free LN and a putative but not yet characterized FN‐proteoglycan complex.
Coexpression of ß1‐ and ß2‐Adrenergic Receptors on Bovine Brain Capillary Endothelial Cells in Culture
Authors: Durieu‐Trautmann O. , Foignant N. , Strosberg A. D. , Couraud P. O.
Published: October 2006

Abstract: The pharmacological study of the blood‐brain barrier has often been hampered by the unavailability of a large number of pure and fully differentiated brain capillary endothelial cells. Here we describe a homogeneous culture of brain capillary endothelial cells isolated from bovine brain (BBECs), which retain at least some phenotypic characteristics of the functional blood‐brain barrier: intercellular tight junctions and monoamine oxidase activity. These cells were subcultured in vitro, in the absence of any neuronal or glial influences, for > 100 doublings without any sign of senescence. The present study is focused on the expression of ß‐adrenergic receptors on BBECs. By Northern blot hybridization, subtype‐specific ligand binding, and cyclic AMP accumulation experiments, we demonstrate that ß1‐ and ß2‐adrenergic receptors are coexpressed (in the respective proportions of 42 and 58%) on BBEC membranes and are functionally coupled to adenylate cyclase. This is the first report documenting a significant number of ß1‐adrenergic receptors on brain capillary endothelial cells. The results are discussed in light of the known noradrenergic innervation of brain capillaries.
Gene Expression in Different Cell Types of Aging Rat Brain
Authors: Venugopal J. , Rao Kalluri Subba
Published: October 2006

Abstract: “Bound’ and “free’ RNA polymerase activities were assessed in the nuclear fraction of cerebral cortical, neuronal, astroglial, and oligodendroglial cells obtained from rats of young, adult, and old ages. Significant decreases in both the bound and free polymerase II activities were noticed in old brain, as compared to adult brain, in neuronal and oligodendroglial nuclei. In astroglia, only the free polymerase II was found to be affected. No effect of aging could be seen on the activity of bound RNA polymerase I + III. The free RNA polymerase I + III activity was increased from adult to old age in neuronal nuclei, but unchanged in oligodendroglial and astroglial nuclei. The age‐dependent reduction in RNA polymerase II was maximum in oligodendroglial cells, whereas it was least, although still significant, in neuronal cells. DNA isolated from old brain was unable to enhance the transcriptional activity when added to chromatin preparations obtained from rat brains of any of the above ages, and the “old’ chromatin was unable to accept even the “young’ DNA as additional exogenous template. It is concluded that the reduced gene expression noticed in old brain nuclei is due to both altered chromatin/DNA structure and inadequate levels of free RNA polymerase II.
Extracellular Events Induced by γ‐Hydroxybutyrate in Striatum: A Microdialysis Study
Authors: Hechler Viviane , Gobaille Serge , Bourguignon Jean‐Jacques , Maitre Michel
Published: October 2006

Abstract: The modification of dopamine release and accumulation induced by γ‐hydroxybutyrate (GHB) was studied using both striatal slices and in vivo microdialysis of caudate‐putamen. GHB inhibited dopamine release for ∼5–10 min in vitro, and this was associated with an accumulation of dopamine in the tissue. Subsequently, there was an increase in dopamine release. In the microdialysis experiments, low doses of GHB inhibited dopamine release, whereas higher doses strongly increased release; the initial decrease seen in slices could not be detected in vivo. Thus, GHB had a biphasic effect on the release of dopamine: An initial decrease in the release of transmitter was followed by an increase. A time‐dependent biphasic effect was observed when GHB was added to brain slices, and a dose‐dependent biphasic effect was seen in dialysate after systemic administration of GHB. Naloxone blocked GHB‐induced dopamine accumulation and release both in vitro and in vivo. GHB also increased the release of opioid‐like substances in the striatum. A specific antagonist of GHB receptors completely blocked both the dopamine response and the release of opioid‐like substances. These data suggest that GHB increases dopamine release via specific receptors that may modulate the activity of opioid interneurons.
Synergistic Interactions Between α1‐ and α2‐Adrenergic Receptors in Activating 3H‐Inositol Phosphate Formation in Primary Glial Cell Cultures
Authors: Wilson Karen M. , Minneman Kenneth P.
Published: October 2006

Abstract: Norepinephrine (NE)‐stimulated 3H‐inositol phosphate (3H‐InsP) formation in primary glial cell cultures is thought to be due to α1‐adrenergic receptor activation. Surprisingly, the α1‐selective agonists phenylephrine and methoxamine showed only 12–21% of the intrinsic activity of NE in activating this response. Although the α2‐selective agonist UK 14,304 was itself inactive, inclusion of UK 14,304 increased the response to the α1‐selective agonists by about threefold. This increase was concentration‐dependent and occurred at all time points examined. 6‐Fluoro‐NE and α‐methyl‐NE mimicked the effect of NE in glial cultures, although with lower potencies. However, several partial agonists were ineffective in activating this response, in both the presence and absence of UK 14,304. Synergistic interactions were not observed for α1‐mediated responses in slices of rat cerebral cortex, either for formation of 3H‐InsPs or potentiation of isoproterenol‐ or adenosine‐stimulated cyclic AMP accumulation. Both UK 14,304 and phenylephrine inhibited NE‐stimulated 3H‐InsP formation in concentrations similar to those necessary to activate this response directly. These results suggest that NE activates 3H‐InsP formation in primary glial cultures by synergistic actions on both α1‐ and α2‐adrenergic receptors. The agonists UK 14,304 and phenylephrine also can act to inhibit the response to NE competitively.
Pharmacological and Biochemical Properties of the γ‐Aminobutyric Acid–Benzodiazepine Receptor Protein from Codfish Brain
Authors: Deng Lynn , Nielsen Mogens , Olsen Richard W.
Published: October 2006

Abstract: The γ‐aminobutyric acidA (GABAA) receptor of codfish brain has been purified to homogeneity and contains a single polypeptide band of 56 kDa molecular mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS‐PAGE) of codfish GABA receptor photoaffinity‐labeled by both [3H]flunitrazepam ([3H]Flu) and [3H]muscimol showed a single radioactive peak with molecular mass of 56 kDa, in contrast to the multiple subunits found in other vertebrate species. The codfish receptor, purified using benzodiazepine (BZ, Ro 7‐1986/1) affinity chromatography, contains an apparent single band both by isoelectric focussing and on a silver‐stained SDS gel. The receptor density and affinity constants for [3H]muscimol and [3H]Flu binding are comparable to those in mammalian brain, and the specific activity (>1,000 pmol/mg of protein) is comparable to that of preparations purified from those sources. The pharmacological specificity of the codfish GABA–BZ receptor is generally similar to that of mammalian brain, including GABA–BZ coupling. The BZ binding exhibits homogeneous kinetic properties resembling those of the mammalian BZ2 receptor type, and shows strong GABA enhancement of [3H]Flu binding and weaker pentobarbital potentiation. This is consistent with other observations of an earlier phylogenetic, as well as ontogenetic, emergence in mammals of the BZ2 receptor subtype than the BZ1. Codfish GABA receptor is postulated to be a homo‐oligomer in which the conformation of GABA and BZ recognition sites is very similar to that in the mammalian hetero‐oligomeric GABAA receptor. The codfish receptor appears to be encoded by an ancestral gene and indicates an early development of BZ–GABA coupling.
Analysis of the Structure and Expression of the Human Dopamine D2A Receptor Gene
Authors: Gandelman Kuan‐Yun , Harmon Steve , Todd Richard D. , O'Malley Karen L.
Published: October 2006

Abstract: In order to study the possible involvement of dopamine receptors in the pathophysiology of various neurological and psychiatric disorders, we have isolated the human D2A gene. Like the rat D2A gene, the human gene contains at least eight exons and spans at least 52 kb. Exons 2–8 are clustered within 14 kb of genome. Exon 1 is separated from exon 2 by at least 38 kb. We and others have shown that alternative utilization of exon 6 gives rise to alternative D2A transcripts. Despite the extreme size of intron 1, no alternative transcription between exons 1 and 2 can be detected in basal ganglia and pituitary using polymerase chain reaction analysis. The relative abundance and tissue distribution of the alternative D2A transcripts were examined in 18 human brain regions. The relative expression of the transcripts varied by at least 70‐fold across the brain regions surveyed. As expected, high levels of transcripts were detected in caudate, putamen, and pituitary. Moderate levels were detected in regions of catecholamine‐containing cell bodies and in the amygdala. In contrast to the rat brain, very low levels of transcripts were detected in cortical regions.
Analysis and Quantitation of the β‐Amyloid Precursor Protein in the Cerebrospinal Fluid of Alzheimer's Disease Patients with a Monoclonal Antibody‐Based Immunoassay
Authors: Henriksson Thomas , Barbour Robin M. , Braa Sonia , Ward Pamela , Fritz Lawrence C. , Johnson‐Wood Kelly , Chung Hyung D. , Burke William , Reinikainen Kari J. , Riekkinen Paavo , Schenk Dale B.
Published: October 2006

Abstract: One of the major clinical findings in Alzheimer's disease (AD) is the formation of deposits of β‐amyloid protein in amyloid plaques, derived from the β‐amyloid precursor protein (β‐APP). To determine the possible use of β‐APP as a diagnostic marker for AD in CSF, a monoclonal antibody‐based immunoassay specific for this protein was developed. The assay does not differentiate between β‐APP695 and β‐APP751 forms but does preferentially recognize β‐APP751 complexed with a protease. Of the two sets of CSF samples tested, one set, obtained from living patients, gave a slightly lower level of β‐APP in AD and Parkinson's disease patients relative to controls, whereas the other set, composed of postmortem samples, showed no significant differences between the AD and control groups.
Preproenkephalin mRNA and Methionine‐Enkephalin Increase in Mouse Striatum After 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Treatment
Authors: Gudehithlu K. P. , Duchemin A. M. , Tejwani G. A. , Neff N. H. , Hadjiconstantinou M.
Published: October 2006

Abstract: Dopaminergic neurons that project to the striatum from the substantia nigra are thought to modulate methionine‐enkephalin (Met‐Enk) metabolism in the striatum. We administered a dose of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) that produces a moderate depletion of dopamine in striatum, about 50%, without overt motor deficits, and found that Met‐Enk‐like immunoreactivity and preproenkephalin mRNA content increased in the tissue. Pretreatment with the monoamine oxidase B inhibitor deprenyl or the dopamine transport blocker nomifensine prevented these changes, suggesting that the changes were related to the partial loss of dopaminergic neurons rather than to MPTP. Moreover, administering GM1 ganglioside, which partially restores the MPTP‐induced dopaminergic deficit, partially corrected the Met‐Enk changes in the striatum as well. These findings are consistent with the hypothesis that dopaminergic input to the striatum, in part, modulates Met‐Enk metabolism. Moreover, they show that moderate nigrostriatal lesions are sufficient to elevate Met‐Enk and preproenkephalin mRNA contents and that restoration of dopaminergic function, as in our studies with GM1 ganglioside, restores the content of Met‐Enk.
Dantrolene Prevents Glutamate Cytotoxicity and Ca2+ Release from Intracellular Stores in Cultured Cerebral Cortical Neurons
Authors: Frandsen Aase , Schousboe Arne
Published: October 2006

Abstract: Using primary cultures of cerebral cortical neurons, it has been demonstrated that the antihyperthermia drug dantrolene completely protects against glutamate‐induced neurotoxicity. Furthermore, in the presence of extracellular calcium, dantrolene reduced the glutamate‐induced increase in the intracellular calcium concentration by 70%. In the absence of extracellular calcium, this glutamate response was completely blocked by dantrolene. Dantrolene did not affect the kinetics of [3H]glutamate binding to membranes prepared from similar cultures. These results indicate that release of calcium from intracellular stores is essential for the propagation of glutamate‐induced neuronal damage. Because it is likely that glutamate is involved in neuronal degeneration associated with ischemia and hypoxia, the present findings might suggest that dantrolene and possibly other drugs affecting intracellular calcium pools might be of therapeutic interest.
12‐Lipoxygenase Products Attenuate the Glutamate Release and Ca2+ Accumulation Evoked by Depolarization of Hippocampal Mossy Fiber Nerve Endings
Authors: Freeman E. J. , Damron D. S. , Terrian D. M. , Dorman R. V.
Published: October 2006

Abstract: Presynaptic correlates of evoked neurotransmitter release include a rise in cytosolic free calcium level and the calcium‐dependent liberation of unesterified arachidonic acid. It has been proposed that lipoxygenase metabolites produced from arachidonic acid may constitute an endogenous feedback system for the modulation of neurotransmitter release. The results of the present study are in agreement with this hypothesis. It was demonstrated that membrane depolarization evoked the release of endogenous glutamate from hippocampal mossy fiber synaptosomes, as well as the accumulation of intraterminal free calcium. The presence of 12‐lipoxygenase products attenuated both the induced release of glutamate and the increase in calcium content, whereas 5‐ or 15‐lipoxygenase metabolites were ineffective. A role for lipoxygenase products in the negative modulation of mossy fiber secretion processes was further indicated by the observations that low concentrations of the lipoxygenase inhibitor nordihydroguaiaretic acid (0.1–10 μM) potentiated the glutamate release and calcium accumulation induced by membrane depolarization. Therefore, we suggest that 12‐lipoxygenase metabolites provide a presynaptic inhibitory signal that limits neurotransmitter release from hippocampal mossy fiber terminals.
Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration
Authors: Tashiro Tomoko , Komiya Yoshiaki
Published: October 2006

Abstract: Changes in solubility and transport rate of cyto‐skeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1–2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with l‐[35S]methionine (experiment II). Labeled proteins in 6‐mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1° Triton at 4°C. When ax‐onal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.
Presynaptic Adenosine A2 and N‐Methyl‐D‐Aspartate Receptors Regulate Dopamine Synthesis in Rat Striatal Synaptosomes
Authors: Chowdhury Muhammad , Fillenz Marianne
Published: October 2006

Abstract: Dopamine synthesis rate and cyclic AMP concentration were measured in synaptosomes prepared from rat striatum. Dopamine synthesis rate was decreased by the addition of either adenosine deaminase or 8‐phenyltheophylline, an adenosine receptor blocker, and was increased by the addition of 2‐chloroadenosine. The addition of L‐glutamate in the absence of adenosine deaminase decreased both dopamine synthesis rate and cyclic AMP concentration; in the presence of adenosine deaminase, glutamate had no effect on basal dopamine synthesis, but enhanced K+‐stimulated synthesis. Both these effects of glutamate were abolished in Ca2+‐free medium or in the presence of 2‐amino‐5‐phosphonovalerate, an N′‐methyl‐D‐aspartate (NMDA) receptor blocker. In Mg2+‐free medium with adenosine deaminase, glutamate enhanced both basal and K+‐stimulated synthesis. These results suggest that dopaminergic terminals have A2. adenosine receptors, whose activation can stimulate dopamine synthesis by a cyclic AMP‐dependent mechanism, and NMDA receptors, which modulate dopamine synthesis by a Ca2+‐dependent mechanism.
Changes in Polyamine Levels in Rat Brain After Systemic Kainic Acid Administration: Relationship to Convulsant Activity and Brain Damage
Authors: de Vera N. , Artigas F. , Serratosa J. , Martínez E.
Published: October 2006

Abstract: We have examined the effects of systemic kainic acid (KA) administration (9 mg/kg, i.p.) on rat behavior, brain damage, and polyamine levels and the action of the specific ornithine decarboxylase inhibitor α‐difluoromethylornithine (DFMO) on these effects. KA elicited convulsant activity in 63% of the animals. In the acute convulsant phase (1–3 h after KA), a rapid decline (−39% at 3 h) of spermidine content in frontal cortex was found. After the acute convulsant phase, levels of hippocampal spermidine and spermine were reduced (−70 and −66%, respectively, at 8 h). A dramatic increase of putrescine content (681, 1,382, and 336% at 8h, 24h, and 9 days, respectively, after KA) was found, associated with histological signs of cortical brain damage (ischemia and necrosis). There was a close relationship between the concentration of putrescine and signs of delayed toxicity (body weight losses) 24 h and 9 days after KA. DFMO partially antagonized the convulsant activity and reduced the increased putrescine levels to ∼50% of values in KA‐treated animals at 24 h but did not change the pattern of histological damage. The role of polyamines in the early and late phases of KA‐induced neurotoxicity is discussed.
Anomalously High Concentrations of Brain Extracellular Uric Acid Detected with Chronically Implanted Probes: Implications for In Vivo Sampling Techniques
Authors: O'Neill Robert D. , Gonzalez‐Mora Jose‐Luis , Boutelle Martyn G. , Ormonde Dymphna E. , Lowry John P. , Duff Adrienne , Fumero Blas , Fillenz Marianne , Mas Manuel
Published: October 2006

Abstract: The height of peak 2, h2, recorded using linear sweep voltammetry with 350‐μm‐diameter carbon paste electrodes in rat striatum was measured from the day of implantation (day 0) to 4 months after surgery. The value of h2was at a minimum on day 0 (0.6 ± 0.2 nA; n = 20), rose sharply to a maximum on day 2 (6.3 ± 0.9 nA; n = 12), and decreased to a stable level by day 7 (3.3 ± 0.7 nA; n = 16), which lasted for 4 months (3.2 ± 0.6 nA; n = 9). These changes were shown by microinfusion of uricase to be due to variations in the concentration of extracellular uric acid, although h2 appears to have a small baseline contribution of ∼0.3 nA from 5‐hydroxyindoleacetic acid. The stable value of h2 recorded under chronic conditions was estimated to correspond to a minimal uric acid concentration of 50 μmol/ L, which represents a 10‐fold increase in the extracellular level of this purine metabolite compared with the initial (acute) value. Very similar results were obtained using a mi‐crodialysis technique that detected uric acid directly. These estimates of striatal uric acid concentration are in marked contrast to those obtained using 40‐μm diameter carbon fiber electrodes, which showed a decrease from the acute preparation to < 1 μmol/L under chronic conditions. Large values of h2 were also recorded with chronically implanted paste electrodes in the hippocampus and frontal cortex. The results suggest that large in vivo probes, such as carbon paste electrodes and dialysis tubes, markedly disturb the neurochemical balance in the extracellular fluid even 1 day following implantation and emphasize the need to develop further small sensors for in vivo neurochemical analysis with minimal perturbation.
Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products
Authors: Okuno Sachiko , Fujisawa Hitoshi
Published: October 2006

Abstract: We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable itabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine‐type catechols) were effective at a concentration as low as 10‐7M both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4‐dihydroxyphenylethylene‐glycol‐type catechols) were ineffective at a concentration of 10‐7M but effective at a concentration of 10‐6M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4‐dihydroxyphenylacetic acid‐type catechols) were ineffective even at a concentration of 1 0‐6M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/ labile form to the inactive/stable form by dopamine was also investigated.
Glutamate Dehydrogenase Reaction as a Source of Glutamic Acid in Synaptosomes
Authors: Yudkoff Marc , Nissim Itzhak , Nelson David , Lin Zhi‐Ping , Erecińska Maria
Published: October 2006

Abstract: The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM15NH4Cl and then utilizing gas chromatography‐mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of I15N]glutamatc and [15N]aspartate from 5 mM15NH4Cl was ∼0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2‐oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mMNH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2‐oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM β‐(±)‐ 2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2‐oxoglutarate. It is concluded: (a) compared with the phosphate‐dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2‐oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.
Preferential Synthesis of Prostaglandin D2 by Neurons and Prostaglandin E2 by Fibroblasts and Nonneuronal Cells in Chick Dorsal Root Ganglia
Authors: Vesin Marie‐Françoise , Barakat‐Walter Ibtissam , Droz Bernard
Published: October 2006

Abstract: To determine the type and the relative amount of prostaglandins (PGs) synthesized by various neural tissues, homogenates of meninges, dorsal root ganglia (DRG) capsules, decapsulated DRG, and unsheathed sciatic nerves were incubated with [1‐14C]arachidonic acid. Homogenates of cultured cells (meningeal cells, fibroblasts, and nonneuronal or neuronal DRG cells) were used to specify the cells producing particular PGs. The highest synthetic capacity was found in fibroblast‐rich tissues (meninges and DRG capsules) and in cultures of meningeal cells or fibroblasts. Two major cyclooxygenase products were formed: [14C]PGE2 and an unusual 14C‐labeled compound, Y. The accumulation of compound Y, corresponding probably to 15‐hydroperoxy PGE2, was completely impaired by addition of exogenous GSH, which conversely enhanced the synthesis of [14C]PGE2and promoted the formation of [14C]PGD2. In contrast, decapsulated DRG or unsheathed sciatic nerves displayed a 10–20 times lower capacity to synthesize PGs than fibroblast‐rich tissues and produced mainly [14C]PGE2 and [14C]PGD2. In this case, [14C]PGE2 or [14C]PGD2 synthesis was neither enhanced nor promoted by addition of exogenous GSH. Neuron‐enriched DRG cell cultures allowed us to specify that [14C]PGD2 is the major prostanoid produced by primary sensory neurons as compared with nonneuronal DRG cells. Because PGD2 synthesis in DRG and more specifically in DRG neurons does not depend on exogenous GSH and differs from PGD2 synthesis in fibroblast‐rich tissues, it is concluded that at least two distinct enzymatic processes contribute to PGD2 formation in the nervous system. Although nonneuronal DRG cells give rise to both PGE2 and PGD2 and fibroblasts produce the largest amount of PGE2, it should be emphasized that PGD2 appears as the prostanoid preferentially, although not exclusively, synthesized by primary sensory neurons.
Skeletal Muscle Proteins Stimulate Cholinergic Differentiation of Human Neuroblastoma Cells
Authors: McManaman James L. , Crawford Frances G.
Published: October 2006

Abstract: Extracts of rat skeletal muscle contain substances that enhance the development of choline acetyltransferase (ChAT) in the cholinergic human neuroblastoma cell line LA‐N‐2. The ChAT enhancing activity in muscle extract was purified to homogeneity by preparative gel electrophoresis and reverse‐phase HPLC. The active factor is biochemically and immunologically identical to ChAT development factor, (CDF), the skeletal muscle factor that enhances ChAT activity in enriched cultures of embryonic rat motoneurons and rescues motoneurons from naturally occurring cell death in vivo. CDF increases the specific ChAT activity of LA‐N‐2 cells fivefold after 6 days in culture, but does not affect their growth or metabolic activity. Basic fibroblast growth factor also increases ChAT activity in LA‐N‐2 cells and its effect is additive with that of CDF. In contrast, neither insulin‐like growth factor‐1, epidermal growth factor, nor nerve growth factor affected the ChAT activity of LA‐N‐2 cells. Our study demonstrates for the first time that CDF can directly affect the development of neuronal properties in a homogeneous population of cells, and that the effects of CDF are separate from those of other types of trophic factors.
Laminar Alterations in γ‐Aminobutyric AcidA, Muscarinic, and β Adrenoceptors and Neuron Degeneration in Cingulate Cortex in Alzheimer's Disease
Authors: Vogt Brent A. , Crino Peter B. , Volicer Ladislav
Published: October 2006

Abstract: The laminar distribution of binding to a number of postsynaptic neurotransmitter receptors was assessed autoradiographically in postmortem samples of area 23a in posterior cingulate cortex from 13 Alzheimer and nine age‐matched control cases. Specific binding in all Alzheimer cases was compared to that in control cases, and the following alterations were observed: reduced muscimol binding in most layers; no changes in pirenzepine binding; and elevated cyanopindolol binding in layers Ic, IIIc, and IV. The Alzheimer cases were classified further on the basis of neuronal degeneration: class 1, no neuron loss; class 2, greatest losses in layer II or HI; class 3, greatest losses in layer IV; and class 4, greatest losses in layer V or VI. This classification uncovered further alterations in ligand binding patterns. First, muscimol binding was reduced in layers II and III only in class 2 cases and in layers V and VI only in class 4 cases. Second, pirenzepine binding was reduced in layers Ic, IIIa‐b, and VI of class 1 cases and layers Va and VI of class 4 cases. In spite of neuron degeneration in classes 2 and 3, there was no change in pirenzepine binding in these classes. Third, elevated cyanopindolol binding occurred in classes 3 and 4, whereas classes 1 and 2 had normal levels of binding. These results suggest that cases of Alzheimer's disease express heterogeneities in neocortical pathology which are reflected in the laminar patterns of binding to postsynaptic receptors. Reductions in muscimol binding to the γ‐aminobutyric acidA receptor had the closest relationship with neuron degeneration, whereas pirenzepine binding appeared to reflect a compensation in muscarinic receptors for changes in neuron densities.
Morphine and Cocaine Exert Common Chronic Actions on Tyrosine Hydroxylase in Dopaminergic Brain Reward Regions
Authors: Beitner‐Johnson Dana , Nestler Eric J.
Published: October 2006

Abstract: We studied levels of tyrosine hydroxylase immunoreactivity and phosphorylation state in the ventral tegmental area (VTA) and nucleus accumbens (NAc) in an effort to understand better the mechanisms by which these brain reward regions are influenced by opiates and cocaine. In the VTA, chronic, but not acute, administration of either morphine or cocaine increased levels of tyrosine hydroxylase immunoreactivity by 30–40%, with no change observed in the relative phosphorylation state of the enzyme. In the NAc, chronic, but not acute, morphine and cocaine treatments decreased the phosphorylation state of tyrosine hydroxylase, without a change in its total amount. In contrast, morphine and cocaine did not regulate tyrosine hydroxylase in the substantia nigra or caudate/putamen, brain regions generally not implicated in drug reward. Morphine and cocaine regulation of tyrosine hydroxylase could represent part of a common biochemical basis of morphine and cocaine addiction and craving.
Conserved Organization of γ‐Aminobutyric AcidAReceptor Genes: Cloning and Analysis of the Chicken β4‐Subunit Gene
Authors: Lasham Annette , Vreugdenhil Erno , Bateson Alan N. , Barnard Eric A. , Darlison Mark G.
Published: October 2006

Abstract: A series of genomic clones containing DNA that encodes the chicken γ‐aminobutyric acidA (GABAA) receptor β4 subunit have been isolated. These have been restriction mapped and partially sequcnced to determine the structural organization and the size of the β4‐subunit gene. This gene, which comprises nine exons, spans more than 65 kb. The organization of the chicken GABAA receptor β4‐subunit gene has been compared to that of the murine GABAA receptor δ‐subunit gene and to those of the genes that encode other members of the ligand‐gated ion‐channel superfamily, namely muscle and neuronal nicotinic acetylcholine receptors (AChRs). Although the positions of the intron/exon boundaries of GABAA receptor sub‐unit genes are seen to be highly conserved, there are significant differences between the genes that encode GABAA receptor and AChR subunits. These results are discussed in relation to the proposal that this superfamily of ligand‐gated ion‐channel receptor genes arose by duplication of an ancestral receptor gene.
Histochemical Study of Aldehyde Dehydrogenase in the Rat CNS
Authors: Zimatkin Sergey M.
Published: October 2006

A quantitative histochemical method was developed to determine aldehyde dehydrogenase (EC 1.2.1.3; ALDH) activity in the CNS. The distribution of ALDH activity in all rat brain and spinal cord regions is described. Among the CNS neuron structures, high enzyme activity was found in receptor and effector neurons, whereas low activity was noted in perikarya of the majority of intermediate neurons, including all aminergic neurons. A positive correlation was demonstrated between the distribution of ALDH activity among rat CNS microregions (our own data) and the density of dopaminergic terminals, dopamine content, and monoamine oxidase activity (literature data) among the same microregions. They may reflect a spatial linkage between ALDH and the predicted sites of natural aldehyde production. Lower enzyme activity was found in phylogenetically younger brain structures. It may explain the differential resistance of CNS structures to ethanol (acetaldehyde). Among the barrier CNS structures, moderate ALDH activity was found in capillaries and surrounding astrocytes and high activity was noted in ependimocytes covering the brain cavities and those of the vascular plexus. This provides realization of the function of ALDH as a brain metabolic barrier for aldehydes.
Molecular Species of Phosphatidylcholine Containing Very Long Chain Fatty Acids in Human Brain: Enrichment in X‐Linked Adrenoleukodystrophy Brain and Diseases of Peroxisome Biogenesis Brain
Authors: Sharp P. , Johnson D. , Poulos A.
Published: October 2006

Abstract: Molecular species of phosphatidylcholine containing unsaturated (i.e., monoenoic and polyenoic) 32‐ to 40‐carbon (very long chain) fatty acids (VLCFA‐PC) are present in normal human brain, the fatty acid composition changing significantly with development. There is a marked increase in the concentration and a change in the polyenoic VLCFA composition of these molecular species in brains of patients with inherited defects in peroxisomal biogenesis [Zellweger's syndrome, neonatal adrenoleukodystrophy (ALD), and infantile Refsum's disease]. In contrast, there is a marked increase in monoenoic VLCFA‐PC in X‐linked ALD whereas molecular species containing polyenoic VLCFA are minor components.
Phosphorylation by Cyclic AMP‐Dependent Protein Kinase Modulates Agonist Binding to the D2 Dopamine Receptor
Authors: Elazar Zvulun , Fuchs Sara
Published: October 2006

Abstract: Phosphorylation of striatal membranes by cyclic AMP‐dependent protein kinase resulted in a reduction in the affinity of the D2 dopamine receptor toward its agonist N‐propylnorapomorphine while the affinity to D2‐specific antagonists remained unchanged. The inhibitory effects observed by phosphorylation and guanine nucleotides on agonist binding to the D2 receptor were additive. The purified D2 dopamine receptor from bovine striatum was specifically phosphorylated by cyclic AMP‐dependent protein kinase with an apparent stoichiometry of 0.7 mol phosphate/mol receptor. The phosphorylated purified D2 receptor also exhibited a reduced agonist binding activity with no change in antagonist binding. The action of cyclic AMP‐dependent protein kinase on both the membrane preparation and the purified D2 receptor was inhibited by a specific inhibitor of the kinase. These data indicate that phosphorylation mediated by cyclic AMP‐dependent protein kinase may represent a physiological pathway for modulation of the receptor binding activity.