bluuebunny/crossref_metadata_2025 huggingface dataset:
Age‐Dependent Striatal Excitotoxic Lesions Produced by the Endogenous Mitochondrial Inhibitor Malonate
Authors: Beal M. Flint , Brouillet Emmanuel , Jenkins Bruce , Henshaw Ross , Rosen Bruce , Hyman Bradley T.
Published: October 2006

Abstract: Intrastriatal injection of malonate, a reversible inhibitor of succinate dehydrogenase (SDH), produced age‐dependent striatal lesions, which were significantly greater in 4‐and 12‐month‐old animals than in 1‐month‐old animals. Both histologic and neurochemical studies showed that the lesions were significantly attenuated by administration of the noncompetitive NMDA receptor antagonist MK‐801. Water‐suppressed chemical shift magnetic resonance imaging showed that malonate produces increased striatal lactate concentrations and striatal lesions on T2‐weighted scans that were attenuated by MK‐801. Neurochemical characterization of the lesions showed significant decreases in markers of medium‐sized spiny neurons (GABA and substance P), whereas a marker of medium‐sized aspiny neurons (somato‐statin) was not different from control values, consistent with an NMDA receptor‐mediated mechanism. The effects of intrastriatal injections of malonate on ATP concentrations were compared with those of the irreversible SDH inhibitor 3‐nitropropionic acid (3‐NP). The ATP depletions following an equimolar injection of malonate were less marked and more transient than those of 3‐NP. These results show that the competitive SDH inhibitor malonate produces more transient and milder bioenergetic defects than 3‐NP, which are associated with selective activation of NMDA receptors. The results strengthen the possibility that a subtle impairment of energy metabolism may play a role in the pathogenesis of Huntington's disease.
Inhibition of Succinate Dehydrogenase by Malonic Acid Produces an “Excitotoxic” Lesion in Rat Striatum
Authors: Greene James G. , Porter Richard H. P. , Eller Robert V. , Greenamyre J‐Timothy
Published: October 2006

Abstract: Excitotoxicity and defects in neuronal energy metabolism have both been implicated in the pathogenesis of neurode‐generative disease. These two mechanisms may be linked through the NMDA receptor, activation of which is dependent on neuronal membrane potential. Because the ability to maintain membrane potential is dependent on neuronal energy metabolism, bioenergetic defects may affect NMDA receptor‐mediated excitotoxicity. We now report that reversible inhibition of succinate dehydrogenase (SDH), an enzyme central to both the tricarboxylic acid cycle and the electron transport chain, produces an “excitotoxic” lesion in rat striatum that can be blocked by the NMDA antagonist MK‐801. Male Sprague‐Dawley rats received intrastriatal stereotaxic injections of the SDH inhibitor malonic acid (1 or 2 μmol) in combination with intraperitoneal injections of vehicle or MK‐801 (5 mg/kg) 30 min before and 210 min after malonic acid. Animals were killed 72 h after surgery, and brains were processed for histology, cytochrome oxidase activity, and [3H]MK‐801 and [3H]AMPA autoradiography. The higher dose of malonic acid (2 μmol) produced large lesions that were markedly attenuated by treatment with MK‐801 (28.1 ± 3.6 vs. 4.7 ± 2.6 mm3; p < 0.001). [3H]MK‐801 and [3H]AMPA binding were reduced in the lesions by 60 and 63%, respectively. One micromole of malonic acid produced smaller lesions that were almost completely blocked by MK‐801 treatment (9.6 ± 1.3 vs. 0.06 ± 0.04 mm3; p < 0.0001). The toxic effects of malonic acid were due specifically to inhibition of SDH inasmuch as co injection of a threefold excess of sgccinate with the malonic acid blocked the striatal lesions (p < 0.002). We conclude that inhibition of SDH can lead to NMDA receptor‐mediated excitotoxic neuronal death. These results have implications for the pathogenesis of and therapy for neurodegenerative diseases.
Agonist Regulation of Gene Expression of Adrenergic Receptors and G Proteins
Authors: Hadcock John R. , Malbon Craig C.
Published: October 2006

Abstract: Study of transmembrane signaling via G proteins has focused to a large extent upon investigations of individual G protein‐linked receptor‐effector systems. Agonist‐induced desensitization and down‐regulation of β‐adrenergic receptors, for example, have been studied extensively and adopted as a general model for G protein‐linked receptor regulation. This review focuses not only on agonist regulation of adrenergic receptor gene expression, but also on how agonists regulate opposing adrenergic receptor‐mediated pathways. This important feature of G protein‐mediated pathways, i.e., cross‐regulation and integration of information among several pathways, will be discussed in the context of what has been learned in the adrenergic receptor‐coupled pathways.
Characterization of a Triacylglycerol Lipase That Liberates Arachidonic Acid from Bovine Chromaffin Cells During Secretion
Authors: Galatioto Leonardo E. , Zahler Peter
Published: October 2006

Abstract: Primary cultures of chromaffin cells from bovine adrenal medullae were used as a model to study lipolytic events during stimulus‐secretion coupling. It has been shown that chromaffin cells liberate arachidonic acid in addition to their main secretion product, the catecholamines. To understand more about the mechanism of arachidonic acid liberation, chromaffin cells were labeled with radioactive arachidonic acid, stimulated, and then analyzed for changes in lipid composition. After stimulation with 10−4M acetylcholine, the radioactivity of triacylglycerols decreased to the same extent that the free arachidonic acid level rose. This finding suggests that in bovine chromaffin cells a stimulation‐dependent triacylglycerol lipase (triacylglycerol hydrolase; EC 3.1.1.3) is involved in arachidonic acid liberation. Further work was performed on detection, characterization, and isolation of this enzyme. Triacylglycerol lipase activity was found in whole cell homogenates and in plasma membrane fractions isolated from adrenal medullary tissue. The plasma membrane lipase showed a pH optimum of 4.3. The apparent Michaelis constant was determined as 3.3 × 10−4 mol/L. Ca2+ did not influence the enzymatic activity. To differentiate the plasma membrane triacylglycerol lipase from the previously described plasma membrane diacylglycerol lipase of chromaffin cells, the influence of RG 80267, a specific diacylglycerol lipase inhibitor, was examined. RG 80267 (50 μM) inhibited the triacylglycerol lipase by only 24%, although diacylglycerol lipase was totally inhibited with only 20 μM RG 80267. The pH optimum of homogenate lipase was broad, lying between 4 and 7. Starting from the soluble fraction of whole cell homogenates, the triacylglycerol lipase was partially purified by ultracentrifugation and size‐exclusion chromatography. The molecular mass of the enzyme as determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was found to be between 47 and 57 kDa.
Effects of Catechol‐O‐Methyltransferase Inhibitors and L‐3,4‐Dihydroxyphenylalanine With or Without Carbidopa on Extracellular Dopamine in Rat Striatum
Authors: Kaakkola Seppo , Wurtman Richard J.
Published: October 2006

Abstract: The effects of two new catechol‐O‐methyltransferase (COMT) inhibitors, OR‐611 and Ro 40‐7592, in combination with L‐3,4‐dihydroxyphenylalanine (L‐dopa) with or without carbidopa on extracellular levels of dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3‐O‐methyldopa (3‐OMD), and 5‐hydroxyindoleacetic acid in rat striatum were studied. A dose of 10 mg/kg i.p. of Ro 40‐7592 alone, in contrast to the same dose of OR‐611, decreased the dialysate level of HVA and increased that of DOPAC; this dose was thus used to differentiate between the effects of central and peripheral COMT inhibition. L‐Dopa (50 mg/kg i.p.) alone slightly increased extracellular levels of DA, DOPAC, and HVA. The effects of L‐dopa were potentiated by carbidopa (50 mg/kg i.p.), and even 3‐OMD levels in dialysate samples became detectable. Both OR‐611 and Ro 40‐7592 significantly further increased the DA and DOPAC efflux from striatum produced by L‐dopa. This increase was more pronounced when carbidopa was added to the treatment. OR‐611 did not modify the effect of L‐dopa or carbidopa/L‐dopa on dialysate HVA levels, whereas Ro 40‐7592 markedly reduced those levels. Both OR‐611 and Ro 40‐7592 very clearly suppressed dialysate 3‐OMD levels produced by carbidopa/L‐dopa. Ro 40‐7592 was more effective than OR‐611 in potentiating the effects of L‐dopa or carbidopa/L‐dopa. These in vivo data show that the new COMT inhibitors markedly inhibit the O‐methylation of L‐dopa and increase its availability to brain, which is reflected as increased DA formation. A significant effect can be achieved even by inhibiting only the peripheral COMT activity. The data suggest that COMT inhibitors may be of clinical importance as adjuncts in the treatment of Parkinson's disease.
Changes in the Ganglioside Long‐Chain Base Composition of Rat Cerebellar Granule Cells During Differentiation and Aging in Culture
Authors: Valsecchi Manuela , Palestini Paola , Chigorno Vanna , Sonnino Sandro , Tettamanti Guide
Published: October 2006

Abstract: Changes in the ganglioside long‐chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed‐phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising ∼8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the “b series” (GD1b, GT1b, and GQ1b) than in the “a series” (GM1 and GD1a). During aging in culture, for 8–22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a.
A Brain‐Specific Protein p25 Is Localized and Associated with Oligodendrocytes, Neuropil, and Fiber‐Like Structures of the CA3 Hippocampal Region in the Rat Brain
Authors: Takahashi Miho , Tomizawa Kayoko , Fujita Shinobu C. , Sato Kazuki , Uchida Tsuneko , Imahori Kazutomo
Published: October 2006

Abstract: Developmental expression and cellular localization of a novel brain‐specific 25‐kDa protein (p25), a substrate of tau protein kinase II, were investigated in the rat brain using polyclonal antibodies raised against peptides synthesized based on the p25 amino acid sequence. By western immunoblotting, p25 was found to be expressed only slightly in the embryonic period; the expression increased from 11 days up to 5 weeks of age, and continued to increase gradually until 1–2 years of age. Immunohistochemistry revealed distinct staining of glial cells in most regions of the central nervous system in the adult rat brain. These positively immunostained cells were especially abundant in the white matter, such as the corpus callosum, cingulum, external capsule, and internal capsule. The glial cells were identified as oligodendrocytes, and the nuclei of the cells remained unstained. Whereas the neuropil in most parts of the brain was immunostained less intensely than glias, the neuropil in the first and second layers of the cerebral cortex and the dentate gyrus was relatively strongly stained. Fiber‐like structures were also stained in the CA3 region of hippocampus.
Basis for Phospholipid Incorporation into Peripheral Nerve Myelin
Authors: Boiron Françoise , Spivack Warren D. , Deshmukh Diwakar S. , Gould Robert M.
Published: October 2006

Abstract: To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline‐, glycerol‐, and ethanolamine‐labeled phospholipids into four subfractions: microsomes, mitochondria, myelin‐like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23–24‐day‐old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin‐containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline‐labeled) accumulated in myelin‐rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol‐labeled) and plasmalogen (ethanolamine‐labeled) accumulated in the myelin‐rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin‐like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P0 (Golgi) and myelin basic proteins (more direct).
Identification of p42 Mitogen‐Activated Protein Kinase as a Tyrosine Kinase Substrate Activated by Maximal Electroconvulsive Shock in Hippocampus
Authors: Baraban Jay M. , Fiore Rachel S. , Sanghera Jasbinder S. , Paddon Harry B. , Pelech Steven L.
Published: October 2006

Abstract: Recent studies have demonstrated that administration of an electroconvulsive shock produces a rapid and transient increase in tyrosyl phosphorylation of a ∼40‐kDa protein in rat brain. Initial characterization of this protein's chromatographic properties indicated that it might be a member of a recently identified family of kinases, referred to as mitogen‐activated protein (MAP) kinases, that are activated by tyrosyl phosphorylation. In the present study, we have used MAP kinase antisera to assess the identity of this protein. We have found that the ∼40‐kDa phosphotyrosine‐containing protein comigrates with p42 MAP kinase (p42mapk) and not with two other 44‐kDa MAP kinase family members detected by these antisera. Western blots of proteins immunoprecipitated with MAP kinase antibodies confirm that p42mapk displays increased tyrosyl phosphorylation after an electroconvulsive stimulus. Chromatographic separation of hippocampal extracts indicates that MAP kinase activity elutes in parallel with p42mapk. Accordingly, these studies identify p42mapk as a tyrosyl kinase substrate that is activated by this stimulus and suggest that this form of MAP kinase may be selectively regulated by neuronal stimulation.
Biochemical Characterization of Two Different Forms of the Substance P Receptor in Rat Submaxillary Gland
Authors: Kage R. , Leeman S. E. , Boyd N. D.
Published: October 2006

Abstract: Studies were designed to examine the basis for the difference in molecular weights of the two proteins detected in membrane preparations of rat submaxillary glands after photolabeling with a radioactive analogue of substance P, 125I‐p‐benzoyl‐L‐phenylalanine8‐substance P. When the two proteins were separated and individually digested with endoglycosidase F, the relative molecular weight of each protein was reduced by ∼ 10,000, indicating that the extent of glycosylation of both proteins is the same. To test whether the difference in their molecular weights can be attributed to a difference in the lengths of the two proteins, photolabeled membranes were treated with carboxypeptidase Y before solubilization to remove from each photolabeled protein the carboxy‐terminal portion that extends beyond the membrane. Only one, albeit diffuse, band was now observed that on subsequent deglycosylation with endoglycosidase F was more clearly seen to be a single band, indicating that differing lengths of peptide chains were cleaved from the two proteins. These results permit the interpretation that the difference in the two forms of the substance P receptor present in rat submaxillary glands is due to differences in the length of their carboxy termini.
Co‐Administration of the D2 Antagonist Pimozide Inhibits Up‐Regulation of Dopamine Release and Uptake Induced by Repeated Cocaine
Authors: Parsons L. H. , Schad C. A. , Justice J. B.
Published: October 2006

Abstract: To investigate the hypothesis that the D2 dopamine (DA) receptor regulates DA uptake, as well as release, in the nucleus accumbens (N ACC), rats were pretreated for 10 days with either the selective D2 antagonist pimozide (1.0 mg/kg, i.p.) or vehicle, followed 3 h later by either cocaine (20 mg/kg, i.p.) or saline. On day 11, a microdialysis method was performed in which various DA concentrations (0, 10, and 20 nM DA) were perfused through the dialysis probe to characterize the diffusion of DA through tissue to and from the microdialysis probe (recovery). This diffusion of DA has been shown to be sensitive to changes in release and uptake. Pimozide pretreatment was shown to attenuate significantly a cocaine‐induced increase in the in vivo recovery of DA (p < 0.01). The in vivo recovery for the vehicle/cocaine group was 47 ± 4%, whereas the in vivo recovery for the pimozide/cocaine group was 31 ± 3%. There was no difference between the pimozide/cocaine and control groups (pimozide/saline, 26 ± 2%; vehicle/saline, 26 ± 3%). In vitro probe calibrations indicated no significant difference in probe efficiencies between groups. These data suggest that the D2 receptor is capable of modulating uptake as well as release of DA in the N ACC of the rat.
In Memoriam: Henry McIlwain 1912–1992
Authors: Bachelard Herman
Published: October 2006

It is with great sadness that the Editors, the Editorìal Board, the Publications Committee, and the Officers and Council of the ISN report the death of Professor Henry McIlwain. Henry was an eminent neurochemist and one of the fathers of the ISN and this Journal.

The Editors
Myelin P0‐Glycoprotein: Predicted Structure and Interactions of Extracellular Domain
Authors: Wells Christopher A. , Saavedra Raul A. , Inouye Hideyo , Kirschner Daniel A.
Published: October 2006

Protein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0 (P0‐ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N‐linked glycosylation. The x‐ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x‐ray diffraction data on the orientation of P0‐ED, and we propose how this immunoglobulin‐like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β‐strand predictions for P0‐ED are virtually superimposable with those of the VH domain of the phosphocholine‐binding immunoglobulin M603 of mouse, which has a similar number of residues as P0‐ED and a structure that has been solved crystallographically. The dimensions of P0‐ED from the space‐filling model, developed using PC‐ based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P0‐ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0‐ED from apposed membranes if the molecules were oriented so that the β‐ strands were approximately perpendicular to the membrane surface. The apposed P0‐EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P0‐ED regions analogous to the complementarity‐determining regions of immunoglobulins can account for the recognition of P0‐ED from apposed membranes in the incompletely swollen state. Two of the faces of P0‐ED that show charge complementarity could account for the homophilic interactions of P0‐ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N‐ linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P0‐ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P0 at the membrane surface. Our model shows how the single immunoglobulin‐like domain of P0 can account for distinct structural states of myelin membrane packing by homophilic interactions.
[125I]RTI‐55 Binding to Cocaine‐Sensitive Dopaminergic and Serotonergic Uptake Sites in the Human Brain
Authors: Little Karley Y. , Kirkman Jacob A. , Carroll F. Ivy , Breese George R. , Duncan Gary E.
Published: October 2006

[125I]RTI‐55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [125I]RTI‐55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [125I]RTI‐55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [125I]RTI‐55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two‐phase processes. A curve‐fitting analysis of the data derived in saturation experiments found a high‐affinity site with KD= 66 ± 35 pM and Smax= 13.2 ± 10.1 pmol/g of tissue and a low‐affinity site with KD= 1.52 ± 0.55 nM and Bmax of 47.5 ± 11‐2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI‐55 > GBR‐12909 > mazindol > WIN 35428 > = methylphenidate > (−)‐cocaine > buproprion > (±)‐amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with KD= 370 ± 84 pM. It appears that [125I]RTI‐55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.
Traumatic Brain Injury‐Induced Excitotoxicity Assessed in a Controlled Cortical Impact Model
Authors: Palmer Alan M. , Marion Donald W. , Botscheller Michelle L. , Swedlow Pamela E. , Styren Scott D. , DeKosky Steven T.
Published: October 2006

Using a controlled cortical impact model of traumatic brain injury (TBI) coupled with tissue microdialysis, interstitial concentrations of aspartate and glutamate (together with serine and glutamine) were assessed in rat frontal cortex. Histological analysis indicated that the severity of injury following severe TBI (depth of deformation = 3.5 mm) was approximately twice that occurring following moderate TBI (depth of deformation = 1.5 mm). Both groups demonstrated significant postinjury maximal increases in excitatory amino acid (EAA) concentration, which were proportional to the severity of injury. The mean ± SEM fold increase in dialysate concentrations of aspartate was 38 ± 13 (n = 5) for moderate TBI and 74 ± 12 (n = 5) for severe TBI. Fold increases in glutamate concentrations were 81 ± 26 and 144 ± 23 for moderate and severe TBI, respectively. Although these increases normalized within 20–30 min following moderate TBI, concentrations of aspartate and glutamate took >60 min to normalize after severe TBI. Changes in levels of nontransmitter amino acids were much smaller. Fold increases for serine concentrations were 4.6 ± 0.6 and 7.6 ± 1.7 in moderate and severe TBI, respectively; glutamine concentrations had similar small fold increases (2.6 ± 0.2 and 4.1 ± 0.6, respectively). Calculation of interstitial concentrations following severe TBI indicated that aspartate and glutamate maximally increased to 123 ± 20 and 414 ± 66 μM, respectively. To determine the extent to which such tissue concentrations of EAAs could contribute to the injury seen in TBI, the EAA receptor agonists N‐methyl‐d‐ aspartate and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid were slowly injected into rat cortex. Remarkably similar histological injuries were produced by this procedure, supporting the notion that TBI is an excitotoxic injury.
Immunochemical Characterization of the β2 Subunit of the GABAA Receptor
Authors: Machu Tina K. , Olsen Richard W. , Browning Michael D.
Published: October 2006

To date three β subunits of the GABAA receptor have been identified in rat brain as a result of cDNA library screening. The β2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying β2 mRNA. To study the β2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315–334 of the intracellular loop of the β2 subunit. The antibody, which had been affinity‐purified, recognized the β2 peptide but did not immunolabel homologous β1 and β3 subunit peptides, indicating that this antibody is specific for the β2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54–58 kDa arid exhibited minor labeling of lower‐molecular‐mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system‐specific labeling of a 55‐kDa band that comigrated with the 55‐kDa band of the purified receptor. Quantitative immunolabeling of this 55‐kDa polypeptide permitted direct determination of the relative amounts of the β2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the β2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.
Regionaland Interspecies Differences in Brain Progesterone Metabolism
Authors: Korneyev A. , Guidotti A. , Costa E.
Published: October 2006

Metabolites of [3H]progesterone were studied in slices prepared from different brain regions of male rat, mouse, and monkey. The major metabolites were 5α‐dihydroprogesterone (5α‐DHP) and 3α,5α‐tetrahydroprogesterone (3α,5α‐THP) in rat brain slices, 5α‐DHP and 20α‐ dihydroprogesterone (20α‐DHP) in mouse brain slices, and 20α‐DHP in monkey brain slices. In rat olfactory bulb slices, 5α‐DHP represented 25.2 ± 3.3% of total radioactivity and 3α,5α‐THP 17.5 ± 2.8%, whereas in rat medulla oblongata slices, 5α‐DHP was 31.3 ± 3.5% and 3α,5α‐ THP 5.4 ± 1.5% of total radioactivity. In slices from other rat brain regions, both metabolites represented 12–20% of total radioactivity.‐The highest metabolite content in mouse brain was also detected in olfactory bulb slices, where 5α‐DHP represented 16.6 ± 4.6% and 20α‐DHP 9.5 ± 2.3% of total radioactivity. In cortical and corpus callosum slices of monkey brain, 26.8 ± 4.4% and 2.4 ± 0.5% of total radioactivity, respectively, were converted to 20α‐DHP, and less than 3% of total radioactivity could be attributed to any of the other metabolites detected. The 3α,α‐THP content in both rat and monkey brain was below 1 nM, but increased in rat brain to 6.7 ± 2.5 nM after electroshock. Endogenous 3α,5α‐THP might play an important role in the regulation of rat behavior through the modulation of GABA action on the GABAA receptor. The significant interspecies differences in the brain progesterone metabolism should be considered in evaluating the functional role of neurosteroids in various species.
Amino Acid Neurotransmitters in Nucleus Tractus Solitarius: An In Vivo Microdialysis Study
Authors: Sved Alan F. , Curtis J. Thomas
Published: October 2006

Amino acid neurotransmitters in the nucleus tractus solitarius (NTS) are thought to play a key role in the mediation of visceral reflexes and glutamate has been proposed as the neurotransmitter of visceral afferent nerves projecting to this region. The present studies sought to characterize the use of in vivo microdialysis to examine extracellular fluid levels of amino acids in the NTS of anesthetized rats. Using a microdialysis probe that was 450 μm in length and a sensitive HPLC assay for amino acids, amino acids could be measured in dialysate samples collected from the NTS. Perfusion of the microdialysis probe with 60 mM K±, to elicit depolarization of nerve terminals in the vicinity of the probe, resulted in increased dialysate fluid levels of aspartate, glutamate, glycine, taurine, and GABA. In contrast, glutamine and tyrosine were decreased and other amino acids were not significantly affected. Prior removal of the ipsilateral nodose ganglion did not alter the K±‐evoked changes in dialysate levels of any of these amino acids. Electrical stimulation of the vagus nerves, using a variety of stimulus parameters, did not significantly alter dialysate levels of glutamate or any of the other amino acids that were measured. Blockade of glutamate uptake with dihydrokainate increased dialysate levels of glutamate, aspartate, and GABA, but in the presence of dihydrokainate vagal stimulation did not alter dialysate levels of these amino acids. The results show that in vivo microdialysis can be used to examine amino acid efflux in the rat NTS and provide further evidence for amino acidergic neural transmission in the NTS. However, these studies fail to support the hypothesis that vagal afferents release glutamate or aspartate.
Inhibition of Glutamate Uptake with l‐trans‐Pyrrolidine‐2,4‐Dicarboxylate Potentiates Glutamate Toxicity in Primary Hippocampal Cultures
Authors: Robinson Michael B. , Djali Sina , Buchhalter Jeffrey R.
Published: October 2006

Sodium‐dependent, high‐affinity glutamate transport is generally assumed to limit the toxicity of glutamate in vivo and in vitro, but there is very little direct evidence to support this hypothesis. In the present study, the effects of the specific uptake inhibitor l‐trans‐pyrrolidine‐2,4‐dicarboxylate on the toxicity and clearance of glutamate were examined in hippocampal neuronal cultures. At a concentration that was not toxic by itself, l‐trans‐pyrrolidine‐2,4‐dicarboxylate increased the toxicity of glutamate approximately fivefold and slowed the clearance of glutamate from the extracellular space. This toxicity was almost completely blocked by the N‐methyl‐d‐aspartate receptor antagonist, d‐2‐amino‐5‐phosphonopentanoate. These studies provide direct evidence that sodium‐dependent, high‐affinity glutamate transport limits glutamate toxicity in vitro.
Epitope Mapping of Form‐Specific and Nonspecific Antibodies to Acetylcholinesterase
Authors: Wasserman Linda , Doctor Bhupendra P. , Gentry Mary K. , Taylor Palmer
Published: October 2006

We have mapped the epitopes to which two monoclonal antibodies against acetylcholinesterase (AChE) from Torpedo californica are directed. One antibody, 2C9, has equivalent affinity for both the 5.6S (amphiphilic) and 11S (hydrophilic) enzyme forms; the other, 4E7, recognizes only the amphiphilic form and has been shown previously to require an N‐linked oligosaccharide residue on the protein. Isolation of cyanogen bromide peptides from the amphiphilic form and assay by a competition ELISA for 2C9 and by a direct binding ELISA for 4E7 identified the same peptide, residues 44–82, as containing epitopes against both antibodies. The epitope for 4E7 includes the oligosaccharide conjugated to Asp59, an N‐linked glycosylation site not present in mouse AChE. A 20‐amino‐acid synthetic peptide, RFRRPEPKKPWSQVWNASTY, representing residues 44–63, was synthesized and found to inhibit completely 2C9 binding to 5.6S enzyme at molar concentrations comparable to those of the cyanogen bromide peptide. It was unreactive with 4E7. Fractionation of the synthetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic peptide at equivalent molar concentrations, whereas the peptide RPEPKKPWSGVWNASTY was as effective as the larger synthetic peptide. The crystal structure of AChE shows the peptide to be on the surface of the molecule as part of a convex hairpin loop starting before the first α‐helix.
pH‐Dependent Binding of Synthetic β‐Amyloid Peptides to Glycosaminoglycans
Authors: Brunden Kurt R. , Richter‐Cook Nancy J. , Chaturvedi Nishith , Frederickson Robert C. A.
Published: October 2006

The seinile plaques found within the cerebral cortex and hippocampus of the Alzheimer disease brain contain β‐amyloid peptide (Aβ) fibrils that are associated with a variety of macromolecular species, including dermatan sulfate proteoglycan and heparan sulfate proteoglycan. The latter has been shown recently to bind tightly to both amyloid precursor protein and A/β, and this binding has been attributed largely to the interaction of the core protein of heparan sulfate proteoglycan with Aβ and its precursor. Here we have examined the ability of synthetic Aβ s to bind to and interact with the glycosaminoglycan moieties of proteoglycans. Aβ(1–28) associates with heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate. The interaction of these sulfated polysaccharides with the amyloid peptide results in the formation of large aggregates that are readily sedimented by centrifugation. The ability of both Aβ(1–28) and Aβ(1–40) to bind glycosaminoglycans is pH‐dependent, with increasing interaction as the pH values fall below neutrality and very little binding at pH 8.0. The pH profile of heparin‐induced aggregation of Aβ(1–28) has a midpoint pH of approximately 6.5, suggesting that one or more histidine residues must be protonated for binding to occur. Analysis of the Aβ sequence reveals a consensus heparin‐binding domain at residues 12–17, and this motif contains histidines at positions 13 and 14 that may be involved in the interaction with glycosaminoglycans. This hypothesis is supported by the following observations: (a) Aβ(13–17) binds tightly to a heparin affinity column at pH 4.0, but not at pH 8.0; and (b) an Aβ(13–17) in which histidine residues 13 and 14 have been replaced with serines does not bind to a heparin column at either pH 8.0 or 4.0. Together, the data indicate that Aβ is capable of binding to the glycosaminoglycan chains of proteoglycans, and such an interaction may be relevant to the etiology and pathology of Alzheimer's disease.
1, 2, 3, 4‐Tetrahydro‐2‐Methyl‐4, 6, 7‐Isoquinolinetriol Depletes Catecholamines in Rat Brain
Authors: Liptrot Janet , Holdup David , Phillipson Oliver
Published: October 2006

1, 2, 3, 4‐Tetrahydro‐2‐methyl‐4, 6, 7‐isoquinolinetriol (TMIQ) was synthesised and tested for activity as a dopamine‐depleting agent in rat brain. After intracerebroventricular infusion, TMIQ caused reductions in dopamine concentrations in substantia nigra, striatum, hypothalamus, and dorsal raphe, and reduction in noradrenaline concentrations in locus coeruleus. TMIQ also reduced 5‐hydroxytryptamine concentrations in dorsal raphe and substantia nigra, although with a lower potency. Comparisons between TMIQ and MPTP showed that they were approximately equipotent in depleting dopamine in the substantia nigra, hypothalamus, and dorsal raphe. Pretreatment of animals with a combination of monoamine oxidase A and B inhibitors completely prevented the TMIQ‐induced reductions in dopamine concentrations in substantia nigra and hypothalamus. Direct unilateral intrastriatal injections of TMIQ produced marked ipsilateral reductions in striatal dopamine, correlating with a behavioural response consisting of turning towards the side of injection. The results suggest that TMIQ should be evaluated further as a possible MPTP‐like compound, which may derive from endogenous β‐hydroxylated catecholamines.
Developmental Regulation of mRNAs Encoding Rat Brain Kainate/AMPA Receptors: A Northern Analysis Study
Authors: Durand Guylaine M. , Zukin R. Suzanne
Published: October 2006

Functionally diverse kainate/α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors are generated by assembly of glutamate receptor (GluR)1, 2, and 3 subunits into homomeric and heteromeric channels. We examined GluR1, 2, and 3 gene expression in embryonic, neonatal, and adult rat brain by northern analysis under conditions of high stringency. In the adult, hybridization to a GluR1 riboprobe revealed the presence of an abundant RNA species, 5.2 kb in size, and minor bands of 3.2 and 3.9 kb. GluR2 hybridized to two species, 3.9 and 5.9 kb, of comparable abundance, presumably attributable to alternate splice products. Hybridization to the GluR3 riboprobe showed a major species of 5.2 kb. This pattern of RNA species was invariant over all the brain regions examined. Examination of GluR expression in development revealed that in the postnatal period, GluR1, 2, and 3 mRNAs are regulated as a function of age. In adult rat brain, GluR1 and 2 mRNA expression was highest in hippocampus; GluR3 was expressed at highest density in hippocampus and frontal cortex. The three transcripts were first detected at embryonic day 16 and then exhibited changes in expression levels in a region‐specific manner. In hippocampus, all three transcripts exhibited elevated expression in the late neonatal period; in frontal cortex, elevated expression was observed for GluR2 and 3 only. In striatum, all three transcripts were expressed at relatively low levels throughout development, with a modest peak at postnatal day 14. In cerebellum, the GluR1 mRNA level was high from postnatal day 28 to adult. Studies of recombinant receptors indicate that GluR1 and 3 subunits form channels that are Ca2± permeable; heteromeric receptors containing the GluR2 subunit are Ca2± impermeable. These observations raise the possibility that a modification of GluR2 expression during development may regulate the level of Ca2±‐permeable kainate channels. We therefore investigated the expression of the GluR2 subunit relative to those of the GluR1 and 3 subunits as a function of age. In hippocampus and frontal cortex, the (GluR1 ± GluR3)/GluR2 ratio declined with increasing age. This result provides evidence for a developmental regulation of kainate/AMPA receptor gene expression in the brain during the first 2 weeks of postnatal life and predicts a decrease in glutamate‐operated Ca2± permeability mediated through kainate/AMPA receptors as a function of age.
Sequence and Novel Distribution of the Chicken Homologue of the Mammalian γ‐Aminobutyric AcidA Receptor γ 1 Subunit
Authors: Glencorse Thora A. , Darlison Mark G. , Barnard Eric A. , Bateson Alan N.
Published: October 2006

cDNAs have been cloned that encode the chicken (Gallus domesticus) γ‐aminobutyric acidA receptor γ1 subunit, the mature sequence of which shares 90, 79, and 69% identity with those of the rat γ1, γ2., and γ3 subunits, respectively. In situ hybridization reveals that there are pronounced differences in the regional and cellular localizations of the corresponding γ‐aminobutyric acidAreceptor γ‐subunit mRNA compared with that of the γ2‐subunit mRNA in 1 ‐day‐old chick brain. The absence of the γ1‐subunit transcript in certain chick brain nuclei of visual and auditory pathways, in which γ 2‐subunit mRNA is present, points to differences in the functional roles of receptors containing one or other of these polypeptides. Certain cells in other brain regions appear to contain both γ1 ‐ and γ2‐subunit mRNAs, suggesting that they either have two γ‐aminobutyric acidA receptor subtypes or possess receptors incorporating two different γ subunits. We have also found contrasts in the distribution patterns, in homologous brain regions, of the chicken γ1‐subunit mRNA and the rat γ1‐subunit mRNA. These data may reflect different functional roles of the chicken and rat γ1subunits.
Functional Identification and Molecular Cloning of a Human Brain Vesicle Monoamine Transporter
Authors: Erickson Jeffrey D. , Eiden Lee E.
Published: October 2006

A vesicle monoamine transporter was functionally identified, molecularly cloned, and characterized from a human substantia nigra cDNA library. The ATP‐dependent transport of 5‐[3H]hydroxytryptamine ([3H]5‐HT) by digitonin‐permeabilized fibroblasts expressing the vesicle monoamine/H± antiporter in culture exhibited a Km of 0.55 μM. Reserpine and tetrabenazine, inhibitors of two monoamine binding sites, effectively blocked [3H]5‐HT accumulation with K1 values of 34 and 78 nM, respectively. Pretreatment of cells with as little as 10 nM reserpine in the presence of ATP abolished uptake. The rank order for substrate inhibition of [3H]5‐HT uptake for both the previously reported rat vMAT1 and the human transporter clone followed the order 5‐HT > dopamine > epinephrine > norepinephrine > 1 ‐methyl‐4‐phen‐ ylpyridinium > 2‐phenylethylamine > histamine. The virtually identical transport characteristics of rvMATI and hvMAT1 confirm the relevance of neuropharmacological studies of rat brain biogenic amine uptake and storage to human brain neurochemistry.
Activation of p42 Mitogen‐Activated Protein Kinase by Glutamate Receptor Stimulation in Rat Primary Cortical Cultures
Authors: Fiore R. S. , Murphy T. H. , Sanghera J. S. , Pelech S. L. , Baraban J. M.
Published: October 2006

Abstract— Recent studies have identified at least two homologous mitogen‐activated protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help define the role of these MAP kinases in neuronal signalling, we have used primary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and by synaptic activity. Regulation was assayed by monitoring changes in both tyrosine phosphorylation on western blots and in vitro kinase activity toward a selective MAP kinase substrate peptide. In initial studies, we found that phorbol ester treatment increased tyrosine phosphorylation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate receptors, i.e., trans‐(±)‐1‐amino‐1,3‐cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)‐α‐(carboxycyclopropyl)glycine. MAP kinase activity and p42 MAP kinase tyrosine phosphorylation were also stimulated by the ionotropic glutamate receptor agonist, kainate, but not by N‐methyl‐d‐aspartate. To examine regulation of MAP kinase by synaptic activity, cultures were treated with picrotoxin, an inhibitor of GABAA receptor‐mediated inhibition that enhances spontaneous excitatory synaptic activity. Treatment of cultures with picrotoxin elicited activation of MAP kinase. This response was blocked by tetrodotoxin, which suppresses synaptic activity. These results demonstrate that p42 MAP kinase is activated by glutamate receptor agonist stimulation and by endogenous synaptic activity.
Age‐Related Regional Changes in Hydroxyl Radical Stress and Antioxidants in Gerbil Brain
Authors: Zhang Jue‐Rong , Andrus Paula K. , Hall Edward D.
Published: October 2006

Abstract— The levels of hydroxyl radicals and oxidized GSH have been examined as indices of oxidative stress in young (3 months), middle‐aged (15 months), and old (20–24 months) gerbil brain hippocampus, cortex, and striaturn. The hydroxyl radical stress was estimated by measuring the salicylate hydroxyl radical trapping products 2,5‐and 2,3‐dihydroxybenzoic acid. The stress was significantly higher in all three brain regions in middle‐aged and old gerbils versus young animals (66.0%). Regional comparisons showed that the stress was significantly higher in cortex than in either the hippocampus or striatum of the middle‐aged and old gerbils (32.0%). The ratio of oxidized to total GSH also increased progressively in middle‐aged and old animals in all three brain regions (p < 0.05, 41.1%), further indicating a general age‐related increase in oxidative stress. Parallel to this age‐related increase in oxidative stress, a significant, albeit slight (8%), decrease in neuronal number in hippocampal CA1 region was observed in both the middle‐aged and old animals. Possible differences in antioxidant levels were also examined. Total GSH levels were similar across age groups (variance <12%). However, the regional comparison showed that it was highest in striatum in all age groups. The levels of a‐tocopherol (vitamin E) were significantly higher in the middle‐aged and old animals in all three regions (70.4%). Vitamin E was highest in the hippocampus and the differences between the hippocampus and the cortex and striatum increased with age. Although of a lesser magnitude, significant increases in hippocampal total ascorbic acid level were also noted with age (p < 0.05, 10%). Ascorbic acid was the most regionally specific of the three antioxidants examined, with hippocampus > cortex > striatum for all age groups. The difference in ascorbic acid level between hippocampus and cortex also increased with age (64.4%). The results suggest that the general age‐related, regionally specific increases in oxidative stress stimulate the accumulation of antioxidants. It is interesting that the hippocampus, which is selectively vulnerable to various insults such as ischemia, epilepsy, and insulin‐induced hypoglycemia, exhibits the greatest age‐related increase in vitamin E and ascorbic acid, perhaps reflective of a greater impact of the progressive increase in baseline oxidative stress.
ATP‐induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors
Authors: Rhoads Allen R. , Parui Rabin , Vu Ngoc‐Diep , Cadogan Robert , Wagner Paul D.
Published: October 2006

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2‐type pur‐inergic receptor. ATP‐induced norepinephrine release was inhibited 80% when extracellular Ca2+ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2‐methylthio‐ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP‐induced secretion was inhibited by Mg2+, and this inhibition was overcome by the addition of excess ATP suggesting that ATP4‐was the active ligand. ATP‐induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12‐O‐tetradecanoylphorbol 13‐acetate. The stimulatory effects of 12‐O‐tetradecanoyl‐phorbol 13‐acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α‐32P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP‐γS and BzATP but not GTP blocked labeling of the proteins by [32P]BzATP. Labeling of the 50‐kDa protein was more sensitive to competition by 2‐methylthio‐ATP than the other labeled proteins, suggesting that the 50‐kDa protein represents the P2 receptor responsible for ATP‐stimulated secretion in these cells.
Maintenance of Neuronal Glutathione by Glial Cells
Authors: Sagara Jun‐ichi , Miura Kenji , Bannai Shiro
Published: October 2006

Abstract— Glutathione levels in neurons and gllal cells were investigated in a neuronal‐glial coculture and in separate cultures. Brain cell suspensions obtained from cerebral hemispheres of fetal rats were cultured, and after 5 days the glutathione content of this cell population, consisting mainly of neurons and astroglial cells, was 23.0 nmol/mg of cell protein, with a significantly high content in glial cells (28.0 nmol/mg of protein) in comparison with neurons (18.8 nmol/mg of protein). When the neurons and glial cells were separated and recultured in fresh medium, neu‐ronal glutathione rapidly decreased, whereas glial glutathione remained unchanged. Cysteine is a rate‐limiting precursor for glutathione synthesis, and its level was also decreased in neurons, but not in glial cells. Cysteine was taken up rapidly by both neurons and glial cells, but cys‐tine was taken up only by glial cells. This accounts for the rapid decrease of glutathione in the cultured neurons, because the culture medium contains cystine, but not cys‐teine. It was also found that the cultured glial cells released cysteine into the medium. These results suggest that neurons maintain their glutathione level by taking up cysteine provided by glial cells.