bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid (AMPA) Binding Sites by Nitric Oxide
Authors: Dev K. K. , Morris B. J.
Published: July 2010

Abstract: Nitric oxide release is reported to be involved in physiological processes associated with altered sensitivity of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) class of glutamate receptor. A series of compounds liberating nitric oxide were therefore tested for their ability to modulate in vitro the characteristics of [3H]AMPA binding to sections of rat brain. Pretreatment of forebrain or cerebellar sections with sodium nitroprusside (1 mM), S‐nitroso‐N‐acetylpenicillamine (SNAP, 200 µM), glyceryl trinitrate (1 µM), or isosorbide dinitrate (0.5 mM) all increased the binding of 3 nM [3H]AMPA by 15–30%. These actions were reproduced by 8‐bromo‐cyclic GMP (200 µM) in the cerebellum but not in the forebrain. In a similar manner, the effect of SNAP was attenuated by an inhibitor of cyclic GMP‐dependent protein kinase in the cerebellum but not in the forebrain. The elevated [3H]AMPA binding observed after pretreatment with SNAP was caused by an increase in binding affinity, but the capacity of the sites was unchanged. Autoradiographic analysis showed that forebrain binding was enhanced in the cerebral cortex and hippocampus but not in the striatum. Nitric oxide therefore appears to be able to increase the affinity of AMPA binding sites via two distinct mechanisms in different brain areas. This action may contribute to synaptic plasticity associated with nitric oxide release.
Regulation of the Release of Interleukin‐6 from Human Astrocytoma Cells
Authors: Cadman Evelyn D. , Witte David G. , Lee Chi‐Ming
Published: July 2010

Abstract: Recent evidence suggests that the level of interleukin‐6 (IL‐6) is elevated in Alzheimer's disease (AD) brains. IL‐6 is produced by reactive glial cells and could potentially affect neuronal survival. Understanding the biochemical mechanism that regulates the production and release of IL‐6 by astrocytic cells may help to identify potential targets for therapeutic intervention in AD. In the present study, glial fibrillary acidic protein‐positive human U373MG astrocytoma cells were used as a model of reactive astrocytes. Production of IL‐6 in response to drug treatment was monitored with an ELISA assay. Histamine (1–100 µM), substance P (SP; 1–100 nM), and human interleukin‐1β (IL‐1β; 1–30 pM) stimulated the release of IL‐6 in a time‐ and concentration‐dependent manner, with EC50 values of 4.5 µM, 8 nM, and 4.5 pM, respectively. The respective effects of histamine, SP, and IL‐1β were effectively blocked by the histamine H1, SP, and IL‐1 receptor antagonists, supporting a receptor‐mediated event for these agents. Both histamine and SP enhanced the formation of inositol phosphates and increase intracellular calcium levels, suggesting that the phosphatidyl‐inositol bisphosphate/protein kinase C pathway may be involved in the IL‐6 release process. Indeed, phorbol 12‐myristate 13‐acetate, a protein kinase C activator, also evoked IL‐6 release from the U373MG cells. On the other hand, IL‐1β, which produces a much more robust release of IL‐6 than histamine or SP, has no effect on inositol phosphate formation or intracellular calcium levels. The biochemical mechanism of the release of IL‐6 in response to IL‐1β remains to be elucidated.
Efficacy of Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 on Neurochemical and Behavioral Deficits Associated with Partial Nigrostriatal Dopamine Lesions
Authors: Altar C. Anthony , Boylan Carolyn B. , Fritsche Michelle , Jones Bruce E. , Jackson Carl , Wiegand Stanley J. , Lindsay Ronald M. , Hyman Carolyn
Published: July 2010

Abstract: Brain‐derived neurotrophic factor (BDNF) promotes the survival of dopamine (DA) neurons, enhances expression of DA neuron characteristics, and protects these cells from 6‐hydroxydopamine (6‐OHDA) toxicity in vitro. We tested the ability of BDNF or neurotrophin‐3 (NT‐3) to exert similar protective effects in vivo during chronic delivery of 6‐OHDA to the rat neostriatum. Chronic infusions of BDNF or NT‐3 (12 µg/day) above the substantia nigra were started 6 days before and continued during an 8‐day chronic intrastriatal infusion of 6‐OHDA. In control and neurotrophin‐treated animals, 6‐OHDA treatment selectively depleted 50–60% of nigrostriatal DA nerve terminals but produced little if any loss of pars compacta DA cell bodies. This partial DA lesion resulted in three rotations per minute toward the lesioned hemisphere after treatment with the DA release‐inducing drug d‐amphetamine. Compared with supranigral infusions of vehicle, BDNF and NT‐3 decreased the number of these ipsiversive rotations by 70 and 48% and increased by 20‐ and 10‐fold, respectively, the number of contraversive rotations observed after amphetamine injection. When challenged with the DA receptor agonist apomorphine, BDNF‐ and NT‐3‐treated animals also exhibited a seven‐ and 3.5‐fold increase in the number of contraversive rotations relative to the vehicle group, respectively. Compared with vehicle, BDNF increased striatal levels of homovanillic acid (HVA; 86%), 3,4‐dihydroxyphenylacetic acid (DOPAC; 42%), and 5‐hydroxyindoleacetic acid (5‐HIAA; 32%) and the HVA/DA (43%) and 5‐HIAA/serotonin (34%) ratios in the DA‐denervated striatum. NT‐3 augmented only striatal 5‐HIAA levels (24%). Neither factor altered the 6‐OHDA‐induced decrease in striatal DA levels or high‐affinity DA uptake and thus did not protect against the destruction of DA terminals and did not alter striatal D1 or D2 ligand binding. Choline, GABA, and glutamate uptake in the striatum were not altered by the lesion or neurotrophin treatment. Thus, BDNF and to a lesser extent NT‐3 reverse rotational behavioral deficits and augment striatal DA and 5‐HT metabolism in a partial DA lesion model.
Identification of Catechol‐Protein Conjugates in Neostriatal Slices Incubated with [3H]Dopamine: Impact of Ascorbic Acid and Glutathione
Authors: Hastings Teresa G. , Zigmond Michael J.
Published: July 2010

Abstract: There is evidence to suggest that degeneration of dopaminergic neurons in Parkinson's disease and certain other conditions results from the action of reactive species generated during the oxidation of dopamine. We, therefore, have begun to explore the conditions under which such reactive species are formed. Tissue slices prepared from rat neostriatum were incubated in a standard Krebs bicarbonate buffer for up to 120 min. In the presence of [3H]dopamine (0.01–100 µM), binding of tritium to the acid‐insoluble protein fraction was detected. Binding was attenuated by the addition of ascorbate (0.085–0.85 mM) or glutathione (0.01–1.0 mM) to the buffer. Acid hydrolysis of the protein revealed the presence of cysteinyl‐dopamine and cysteinyl‐dihydroxyphenylacetic acid residues. These results suggest that dopamine oxidizes to form reactive metabolites, presumably quinones, that then bind to nucleophilic sulfhydryl groups on protein cysteinyl residues. The findings further suggest that the extent to which reactive metabolites are formed is determined in part by the balance between the availability of dopamine and the antioxidant environment.
Potassium and Taurine Release Are Highly Correlated with Regulatory Volume Decrease in Neonatal Primary Rat Astrocyte Cultures
Authors: Vitarella Domenico , DiRisio Darryl J. , Kimelberg Harold K. , Aschner Michael
Published: July 2010

Abstract: Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on‐line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K+ (using 86Rb), taurine, and d‐aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 mM quinine, 10 µM nimodipine, 100 µM BAPTA‐AM, 10 µM trifluoperazine, or a calcium‐free buffer significantly (p < 0.0001) inhibited RVD. This was accompanied by inhibition of 86Rb release but an increase in d‐[3H]‐aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium‐activated K+ channels, and inhibition of calmodulin all inhibit RVD. Because d‐[3H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that d‐aspartate release is a main determinant of RVD. In contrast, [3H]taurine release was increased by 1 mM quinine and inhibited by 10 µM trifluoperazine. The net release of K+ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K+ and taurine release in RVD.
Rapid Translocation of Cytosolic Ca2+/Calmodulin‐Dependent Protein Kinase II into Postsynaptic Density After Decapitation
Authors: Suzuki Tatsuo , Okumura‐Noji Kuniko , Tanaka Ryo , Tada Toyohiro
Published: July 2010

Abstract: The postsynaptic density (PSD) fraction prepared from rat forebrains frozen with liquid nitrogen immediately after dissection (within 30 s after decapitation) contained major postsynaptic density protein (mPSDp), α subunit of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) at a level of merely 2.7% of the total protein. The content of the protein in the fraction was increased to ∼10% by placing the forebrains on ice for a few minutes. Accumulation, but to a lesser extent, of the protein after placement was also observed in the particulate, synaptosome, and synaptic plasma membrane fractions with its concomitant decrease in the cytosolic fraction. The distribution change may be translocation of the protein, because the amounts of the losses of the protein in the cytosolic fraction were balanced by the gains in the particulate fractions. By translocation, CaMKII became Triton X‐100 insoluble and partially inactivated. The amount of CaMKII transferred from the cytosol to particulate fractions at 0°C was about the same as that contained in the conventional PSD fraction. Furthermore, the thickness of the PSD was increased by the treatment of the forebrains at 37°C, by which the content of CaMKIIα in the PSD fraction was increased to twofold. These results suggest that most of the CaMKII α subunit associated with the PSD fraction (mPSDp) is translocated from cytosol after decapitation. We also showed similar translocation of CaMKIIβ/β′.
Choline‐Containing Compounds in Human Astrocytomas Studied by 1H NMR Spectroscopy In Vivo and In Vitro
Authors: Usenius Jussi‐Pekka , Vainio Pauli , Hernesniemi Juha , Kauppinen Risto A.
Published: July 2010

Abstract: We have studied 14 patients with different grades of astrocytomas using 1H NMR spectroscopy in vivo. Typically, astrocytomas exhibited a low N‐acetyl‐aspartate peak, a prominent signal from choline group‐containing compounds, and lactate in the 1H NMR spectra in vivo. The uncorrected choline/creatine + phosphocreatine peak area ratios were higher in tumors than in normal brain tissue. Absolute concentration of choline‐containing compounds (1.74 ± 0.09 mmol/L) in the normal brain tissue was not different in any grade of astrocytoma, but total creatine concentration in healthy brain (7.49 ± 0.30 mmol/L) was higher than that in grade IV astrocytomas (4.84 ± 0.89 mmol/L). Relaxation constants of choline‐containing compounds did not differ in tumors from those determined in normal brain. Perchloric acid extracts of biopsy samples from 35 astrocytomas and 13 samples of normal temporal white matter were analyzed with 1H NMR. Total concentration of choline‐containing compounds did not differ between controls and any grade of astrocytoma when the quantification was done in vitro. It is interesting that phosphorylcholine concentration was about twofold greater in grade IV astrocytomas than in controls or other grades of astrocytomas. We conclude that high phosphorylcholine in grade IV astrocytomas may be an indicator of degree of malignancy. The proportional changes within the group of choline‐containing compounds observed in vitro were not reflected in the NMR properties of choline signal in vivo.
Complete Nucleotide Sequence and Tissue‐Specific Expression of the Rat Phenylethanolamine N‐Methyltransferase Gene
Authors: Suh Yoo‐Hun , Chun Yhang‐Sook , Lee In Sik , Kim Sung‐Soo , Choi Woong , Chong Young Hae , Hong Lyhna , Kim Seong‐Hun , Park Chan‐Woong , Kim Chong‐Gook
Published: July 2010

Abstract: The rat phenylethanolamine N‐methyltransferase (PNMT) gene was isolated from a genomic library by cross‐hybridization with a bovine PNMT cDNA probe. Complete nucleotide sequence analysis of a genomic clone showed that this gene contained three exons and spanned about 2.8 kb in length. There were the acute‐phase response element, TATA, SP1, and GRE sequences. The physicochemical properties of rat adrenal PNMT were different from those of the brainstem PNMT. However, northern blot and reverse transcription‐polymerase chain reaction analysis showed that the rat PNMT gene may not express the multiple forms of mRNA. These results suggest that the rat PNMT gene might produce a single enzyme protein, whose activity may be differentially modulated by tissue‐specific environment in the central and peripheral systems.
Biochemical and Cellular Properties of Three Immortalized Schwann Cell Lines Expressing Different Levels of the Myelin‐Associated Glycoprotein
Authors: Toda Kenichi , Small Judy A. , Goda Shuichiro , Quarles Richard H.
Published: July 2010

Abstract: Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin‐associated glycoprotein (MAG) were compared. The S16 line generated by repetitive passaging was described previously and expresses a level of MAG comparable to that in adult sciatic nerve. The S42 line was generated independently by the same procedure, divides more slowly than the S16 line, and expresses an even higher level of MAG. The S16Y line arose spontaneously from a passage of the S16 cells, divides much more rapidly, and does not express MAG. The levels of MAG expression in the three lines are inversely related to their rates of proliferation, and MAG mRNA levels parallel the amounts of MAG. The S16 and S42 lines consist mainly of flat cells at low density and develop many processes at high density, whereas most of the S16Y cells are spindle‐shaped, resembling primary Schwann cells in appearance. Surface immunostaining with the O4 antibody was positive for the S16 and S42 cells and negative for the S16Y cells, but all three lines were negative for surface staining with the O1 antibody. The overall protein compositions of the three lines are very similar, but the S16 and S42 cells express larger amounts of several glycoproteins than the S16Y cells, including the adhesion proteins, neural cell adhesion molecule, L1, and laminin. S16 and S42 cells (but not S16Y cells) also express P0 glycoprotein, galactocerebroside, and sulfatide, but, unlike MAG, these other myelin‐related components were present at much lower levels than in adult nerve. Myelin basic protein and proteolipid protein were not detected in any of the lines, although all three lines contained proteolipid protein mRNA. 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase and glial fibrillary acidic protein were present in all three lines. Conditions have not yet been found in which any of the lines will myelinate dorsal root ganglion neurons in vitro, but the S16 and S42 cells differ from the S16Y cells by clustering around neurons after 1 week in coculture. In many respects, the S16 and S42 cells biochemically resemble Schwann cells at an early stage in their preparation to myelinate and should be useful for investigating the cell biology of MAG and other myelin‐related components.
Estrogen Receptor Is Expressed in Different Types of Glial Cells in Culture
Authors: Santagati Sabrina , Melcangi Roberto C. , Celotti Fabio , Martini Luciano , Maggi Adriana
Published: July 2010

Abstract: Estrogens derived from the aromatization of androgens are believed to be responsible for the induction of the sexual differentiation of the CNS interacting with specific estrogen receptors (ER) present in developing neurons. However, the brain cellular distribution of ER is not so well documented. The aim of this study was to investigate the qualitative and quantitative expression of ER mRNA in well characterized cultures of rat type 1 and type 2 astrocytes and of oligodendrocytes by polymerase chain reaction. A series of amplifications with a set of primers spanning along the entire ER mRNA was utilized in the different types of glial cells, in a positive control (uterus), and in a negative control (SK‐N‐BE cell line) previously shown to be devoid of ER. The data obtained show that ER mRNA is expressed in all three types of glial cell analyzed in almost equal amounts, which are 25–50 times lower than those in the uterus. The mRNA expressed in the glia is homologous with that expressed in the uterine tissue.
Dopaminergic Regulation of Septohippocampal Cholinergic Neurons
Authors: Day Jamie C. , Fibiger Hans C.
Published: July 2010

Abstract: The extent to which acetylcholine (ACh) release in the hippocampus is regulated by dopaminergic mechanisms was assessed using in vivo microdialysis in freely moving rats. Systemic administration of the dopamine (DA) receptor agonist apomorphine (1.0 mg/kg) or the specific D1 agonist CY 208–243 (1.0 mg/kg) increased microdialysate concentrations of ACh in the hippocampus. The D2 receptor agonist quinpirole (0.5 mg/kg) produced a small but statistically significant decrease in hippocampal ACh release. d‐Amphetamine (2.0 mg/kg) increased ACh release, an effect that was blocked by the D1 receptor antagonist SCH 23390 (0.3 mg/kg) but not by the D2 antagonist raclopride (1.0 mg/kg). These findings suggest that endogenous DA stimulates septo‐hippocampal cholinergic neurons primarily via actions at D1 receptors. In addition, these results are similar to previous findings regarding the dopaminergic regulation of cortical ACh release, and suggest that the anatomical continuum formed by basal forebrain cholinergic neurons that project to the cortex and hippocampus acts as a functional unit, at least with respect to its regulation by DA.
Inhibitory Effects of Nitric Oxide on the Uptake of [3H]Dopamine and [3H]Glutamate by Striatal Synaptosomes
Authors: Lonart Gyorgy , Johnson Kenneth M.
Published: July 2010

Abstract: Ten‐minute incubation with 1 mMS‐nitroso‐l‐cysteine (NO‐CYS), a nitric oxide (NO) generator, induced a 3.5‐fold increase in the basal dopamine (DA) efflux from rat striatal slices. Reduced hemoglobin (100 µM), a NO scavenger, blocked this response. Nomifensine (10 µM), an inhibitor of high‐affinity DA transport, reduced the NO‐CYS effect by 39%. In a synaptosomal preparation, NO‐CYS induced a concentration‐dependent efflux of [3H]DA that was also significantly inhibited by 10 µM nomifensine. Because these findings indicated that NO could alter the activity of the striatal DA transporter, we tested the effect of NO and NO‐CYS on the uptake of [3H]DA into crude striatal synaptosomes. Although both compounds inhibited [3H]DA uptake with similar dose‐response characteristics (IC50≈300 µM and ≈400 µM, respectively), the effect of NO was quicker in onset. The presence of superoxide dismutase (30 U/ml) and catalase (50 U/ml) had a small impact on the NO‐CYS inhibition of [3H]DA uptake (1.8‐fold increase in IC50). NO (1 mM) inhibited striatal [3H]glutamate uptake by 23%, but lower concentrations had no significant effect. The duration of the effect of NO gas on [3H]DA uptake varied from <5 min to>10 min, depending on the concentration used. All concentrations of NO‐CYS tested produced an inhibition of [3H]DA uptake that lasted at least 10 min. However, only concentrations ≤1 mM NO‐CYS (or NO) were washable. The effect of 3 mM NO‐CYS lasted>20 min and could not be reversed by washing. Reduced hemoglobin (300 µM) prevented the action of 3 mM NO‐CYS, whereas methemoglobin had no effect. The action of 3 mM NO‐CYS was temperature dependent and took about 2 min to fully develop. Scatchard analysis revealed that NO‐CYS diminished the capacity of the DA transporter without having a significant effect on the affinity. NO‐CYS had no effect on striatal [3H]‐mazindol binding, suggesting that NO‐CYS did not interact with the DA recognition site of the transporter. These data suggest that NO may play a role in the regulation of DA and, to a lesser extent, glutamate transport in the striatum.
Production and Metabolism of Platelet‐Activating Factor in the Normal and Ischemic Fetal Rat Brain
Authors: Kunievsky Baruch , Yavin Ephraim
Published: July 2010

Abstract: Production and metabolism of platelet‐activating factor (PAF) in the fetal rat brain under normal and under ischemic stress conditions were examined. Endogenous PAF levels, determined by a bioassay using PAF‐stimulated platelet release of [3H]serotonin, averaged 2.32 ± 2.14 pg/mg in control brains and was reduced to 1.10 ± 1.06 pg/mg after 20 min of maternal‐fetal blood flow occlusion. [3H]PAF administered intracranially into the fetuses in utero was removed in a biphasic, time‐dependent manner: a rapid component with an estimated elimination rate constant of 0.067 min−1 and t1/2 of 10 min and a slower component with an elimination rate of 0.017 min−1 and t1/2 of 41 min. In fetal brains subjected to ischemia a delayed elimination of [3H]PAF was noticed in the slow component (t1/2 = 59 min), indicating a possible difference between the clearance of exogenous and endogenous PAF. The disappearance of [3H]PAF was accompanied by an increase in the radioactivity associated with lyso‐PAF that reached a plateau after 2.5 min, possibly indicating the degradation of the fast component. A steady increase in the alkyl‐acyl‐glycerophosphorylcholine radioactivity commenced after 5 min and continued up to 30 min. The endogenous production of PAF and the rapid degradation due to maternal‐fetal blood flow occlusion indicate an additional target for therapeutic intervention in the pathology of intrauterine ischemia. Addition of the calcium ionophore A23187 stimulated in vitro formation of PAF and lyso‐PAF from [3H]‐choline‐labeled fetal brain phospholipids, suggesting that intracellular calcium may play a major stimulatory role in PAF production. Degradation of polyphosphoinositides by a phospholipase C may constitute a major target for PAF generated either by decapitation or after blood flow occlusion, as evident from the protective effect of the in vivo administered BN52021 PAF antagonist.
Origin of Ischemia‐Induced Glutamate Efflux in the CA1 Field of the Gerbil Hippocampus: An In Vivo Brain Microdialysis Study
Authors: Mitani Akira , Andou Yasushi , Matsuda Seiji , Arai Tatsuru , Sakanaka Masahiro , Kataoka Kiyoshi
Published: July 2010

Abstract: In vivo brain microdialysis experiments were performed in the gerbil to evaluate the origin of accumulation of extracellular glutamate under transient ischemia. Microdialysis probes were positioned in the CA1 field of the hippocampus in which proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals had been induced by 5‐min ischemia 10–14 days before the microdialysis experiment; in the white matter of the cerebral cortex, which contained few neurons, few presynaptic terminals, and many astrocytes; or in the histologically normal CA1 field of the hippocampus, and then 5‐ or 20‐min ischemia was induced. When 5‐min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter of the cerebral cortex, whereas a significant increase in glutamate (15‐fold) was observed in the histologically normal CA1 field. When 20‐min ischemia was induced, no significant increase in glutamate content was observed in the CA1 field that showed proliferation of astrocytes, death of CA1 pyramidal neurons, and damage of presynaptic terminals and in the white matter during the first 10 min after the onset of 20‐min ischemia, but remarkable ischemia‐induced increases in glutamate were observed during the last 10 min of 20‐min ischemia in both areas. An excessive increase in glutamate (100‐fold) was observed during 20‐min ischemia in the normal CA1 field of the hippocampus. When a probe was positioned in the CA1 field of the hippocampus in which presynaptic terminals of Schaffer collaterals and commissural fibers had been eliminated by bilateral kainate injections into the lateral ventricles 4–7 days before the microdialysis experiment and then 5‐min ischemia was induced, a significant increase in glutamate was observed during the last half of 5‐min ischemia. These results suggest that the efflux of glutamate from astrocytes does not contribute to the large ischemia‐induced glutamate accumulation in the CA1 field of the hippocampus during 5‐min ischemia but contributes to the ischemia‐induced increase in glutamate level during ischemia with a longer duration and that ischemia‐induced efflux of glutamate in the CA1 field during 5‐min ischemia originates mainly from neuronal elements: presynaptic terminals and postsynaptic neurons.
Extracellular 5‐Hydroxytryptamine in Median Raphe Nucleus of the Conscious Rat Is Decreased by Nanomolar Concentrations of 8‐Hydroxy‐2‐(Di‐n‐Propylamino)tetralin and Is Sensitive to Tetrodotoxin
Authors: Bosker Fokko , Klompmakers Andre , Westenberg Herman
Published: July 2010

Abstract: Extracellular 5‐hydroxytryptamine (5‐HT) in the median raphe and dorsal hippocampus was measured using in vivo microdialysis. Administration of 60 mM K+ through the probe into the median raphe region significantly increased 5‐HT output from the median raphe and the right dorsal hippocampus. Local infusion of 10 µM tetrodotoxin into the median raphe region substantially decreased 5‐HT in the median raphe and left and right dorsal hippocampus. Systemic administration (0.3 mg/kg s.c.) of 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT) decreased the 5‐HT levels in the dialysates from both the median raphe region and dorsal hippocampus. Administration of 30 nM 8‐OH‐DPAT through the dialysis probe into the median raphe region decreased 5‐HT output from the median raphe and dorsal hippocampus significantly, whereas at concentrations from 60 nM to 10 µM, no significant effects were found in either region. With 100 µM 8‐OH‐DPAT, a significant increase was seen in the median raphe region, but not in dorsal hippocampus. Similar findings were obtained following microinjections of different doses of the compound into the median raphe region. The results of this study indicate that the somatodendritic release of 5‐HT is impulse flow‐dependent. Moreover, the decrease of 5‐HT in the median raphe region by low nanomolar concentrations of 8‐OH‐DPAT supports the notion that somatodendritic 5‐HT release is subject to a local negative feedback mechanism through 5‐HT1A autoreceptors.
Paralytic Tremor (pt): A New Allele of the Proteolipid Protein Gene in Rabbits
Authors: Tosic Mirjana , Dolivo Michel , Domańska‐Janik Krystyna , Matthieu Jean‐Marie
Published: July 2010

Abstract: Paralytic tremor (pt) is a sex‐linked mutation in rabbit that affects myelination of the CNS. Myelin in the pt brains represents ∼30% of the normal levels. Previously we showed that the pt mutation affects primarily proteolipid protein (Plp) gene expression. In the present study we investigated the relative effect of the pt mutation on two distinctive Plp gene products, PLP‐ and DM‐20‐specific messenger RNAs. Our results showed that both PLP and DM‐20 are affected and that the ratio DM‐20/PLP was higher in pt rabbits than in age‐matched controls. We sequenced normal rabbit PLP cDNA and characterized pt mutation at the DNA level. Rabbit PLP sequence, deduced from cDNA, differs from the human protein only at Thr198. Sequence analysis of the mutant cDNA revealed a transversion T → A in exon 2 of the Plp gene. This point mutation, which is placed at the end of the first potential transmembrane domain, results in a substitution of His36 by a glutamine. This transversion abolishes a restriction site that enabled us to screen a large number of animals and observe a perfect correlation between the pt allele and the abnormal phenotype.
τ Phosphorylation in Human, Primate, and Rat Brain: Evidence that a Pool of τ Is Highly Phosphorylated In Vivo and Is Rapidly Dephosphorylated In Vitro
Authors: Garver Timothy D. , Harris Katherine A. , Lehman Ralph A. W. , Lee Virginia M.‐Y. , Trojanowski John Q. , Billingsley Melvin L.
Published: July 2010

Abstract: The extent of τ phosphorylation is thought to regulate the binding of τ to microtubules: Highly phosphorylated τ does not bind to tubules, whereas dephosphorylated τ can bind to microtubules. It is interesting that the extent of τ phosphorylation in vivo has not been accurately determined. τ was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that τ isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope‐specific monoclonal and polyclonal antibodies. This highly phosphorylated τ was dephosphorylated during a 120‐min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow‐mobility forms of τ were shifted to faster‐mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of τ in slow‐mobility, highly phosphorylated forms. Approximately 25% of immunoreactive τ was present as slow‐mobility (66‐ and 68‐kDa) forms of τ. The percentage of immunoreactive τ in faster‐mobility pools (42–54 kDa) increased in proportion to the decrease in content of 66–68‐kDa τ as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on τ is rapid and depends on neuronal phosphatases. Furthermore, τ is highly phosphorylated in normal‐appearing human, primate, and rodent brain. The presence of a highly phosphorylated pool of τ in adult brain may modify the present hypotheses on how paired helical filaments of Alzheimer's disease are formed.
Benzodiazepine Treatment Causes Uncoupling of Recombinant GABAA Receptors Expressed in Stably Transfected Cells
Authors: Klein Ronald L. , Whiting Paul J. , Harris R. Adron
Published: July 2010

Abstract: GABAA and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABAA receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk− cells stably transfected with GABAA receptors (bovine α1, β1, and γ2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [3H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the KD and Bmax of [3H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t1/2 of ∼30 min. The EC50 for clonazepam treatment was ∼0.3 µM, and cotreatment with the benzodiazepine antagonist Ro 15‐1788 (5.6 µM) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents posttranslational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as benzodiazepine tolerance.
Apoptotic Cell Death of a Hybrid Motoneuron Cell Line Induced by Immunoglobulins from Patients with Amyotrophic Lateral Sclerosis
Authors: Alexianu Maria E. , Mohamed A. Habib , Smith R. Glenn , Colom Luis V. , Appel Stanley H.
Published: July 2010

Abstract: Apoptotic cell death has recently been implicated in diseases involving nonproliferating, terminally differentiated cells such as neurons. Previous experiments have documented that immunoglobulins from patients with amyotrophic lateral sclerosis (ALS) can kill motoneuron‐neuroblastoma hybrid cells [ventral spinal cord 4.1 (VSC 4.1)] by a calcium‐dependent process. Here, we studied the mechanism of ALS IgG‐induced cell death. In the presence of ALS IgG the VSC 4.1 cells undergo cell shrinkage and membrane blebbing, which are morphological features of apoptotic cell death. The damaged cells can be identified by in situ end labeling of nicked DNA and biochemically show laddering on agarose gel electrophoresis. This ALS IgG‐triggered process is prevented by cycloheximide, aurintricarboxylic acid, and zinc sulfate. These data demonstrate that immunoglobulins from patients with ALS are able to induce apoptosis in motoneuron hybrid cells and provide a potential mechanism for motoneuron degeneration in human ALS.
Isolation and Expression of an Arrestin cDNA from the Horseshoe Crab Lateral Eye
Authors: Smith W. Clay , Greenberg Robert M. , Calman Bruce G. , Hendrix Miyono M. , Hutchinson Leanna , Donoso Larry A. , Battelle Barbara‐Anne
Published: July 2010

Abstract: Electrophysiological studies of photoreceptors from the horseshoe crab Limulus polyphemus continue to provide fundamental new knowledge of the photoresponse in invertebrates. Therefore, it is of particular interest to characterize the molecular components of the photoresponse in this system. Here we describe an arrestin cloned from a cDNA library constructed using poly(A)+ RNA isolated from Limulus lateral eyes. The protein, deduced from the arrestin cDNA, is most similar to arrestin from locust antennae (56% identity) and Drosophila phosrestin I (53% identity). Limulus arrestin was expressed in a heterologous system, and its properties were compared with those of a 46‐kDa light‐regulated phosprotein (pp46A) in Limulus photoreceptors described in previous studies from this laboratory. Arrestin and pp46A (a) have the same apparent molecular weight on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, (b) have an isoelectric point in the basic pH range, (c) require calmodulin and elevated Ca2+ levels for phosphorylation, (d) are immunoreactive with monoclonal antibody C10C10 directed against a sequence in bovine arrestin (S‐antigen) that is perfectly conserved in the deduced arrestin protein, and (e) are associated with photoreceptors. We conclude that the arrestin described here and pp46A are the same protein. The results of this and previous studies show that in Limulus photoreceptors, light regulates the phosphorylation of arrestin in complex ways.
Mutagenesis of Rat Dopamine β‐Hydroxylase: Examination in Cell‐Free System
Authors: Feng Zhehui , Sabban Esther L.
Published: July 2010

Abstract: Dopamine β‐hydroxylase (DBH; EC 1.14.17.1) exists as membrane‐bound and soluble forms in neurosecretory vesicles. The features of DBH required for glycosylation and incorporation into membranes were studied in a cell‐free system. Translation of full‐length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl‐terminal, in contrast to amino‐terminal, truncations gave translation products that were glycosylated by microsomal membranes. Site‐directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavage site modified. Translation without membranes indicated that the second AUG is not used to initiate translation. The mutant with Glu41→ Leu41 and Ser43→ Thr43 yielded only the GH form with membranes, whereas mutation of Ser43→ Ala43 generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membranes on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moiety. The results suggest a role for cleavage of a signal sequence in the formation of different forms of DBH. The possibility that these mutations change the secondary structure near the signal cleavage site, affecting processing, is discussed.
A Negative Regulatory Element in the Rat Dopamine β‐Hydroxylase Gene Contributes to the Cell Type Specificity of Expression
Authors: Shaskus John , Zellmer Eustacia , Lewis Elaine J.
Published: July 2010

Abstract: Dopamine β‐hydroxylase (DBH) catalyzes the synthesis of the neurohormone norepinephrine and is expressed only in noradrenergic and adrenergic cells of the nervous and endocrine systems. We have previously described a positive‐acting genetic regulatory element of the DBH that contributes to the restricted expression of this gene. In the study described here, we identify and characterize a negative‐acting regulatory element within the 5′‐flanking region of the rat DBH gene. This negative regulatory element, which is located between −282 and −232, represses transcription from the DBH promoter in cell lines of noncatecholaminergic origin and binds to nuclear factors found in extracts from both the catecholaminergic cell line SHSY‐5Y and the noncatecholaminergic cell lines JEG‐3 and C6. The negative regulatory region will reduce transcription from a heterologous promoter in two noncatecholaminergic cell lines. These experiments demonstrate that the selective expression of the DBH gene is controlled by both positive‐ and negative‐acting genetic regulatory elements.
The Dendritic Peptide Neurogranin Can Regulate a Calmodulin‐Dependent Target
Authors: Martzen Mark R. , Slemmon J. Randall
Published: July 2010

Abstract: Neurogranin, a peptide capable of binding the calcium‐poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin‐dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid‐soluble calf brain proteins by a combination of calmodulin‐Sepharose affinity chromatography and reverse‐phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration‐dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium‐dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to ∼1 µM calcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C‐phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin‐requiring targets in neurons.
Activation of Metabotropic Glutamate Receptors Prevents Neuronal Apoptosis in Culture
Authors: Copani Agata , Bruno Valeria M. G. , Barresi Vincenza , Battaglia Giuseppe , Condorelli Daniele F. , Nicoletti Ferdinando
Published: July 2010

Abstract: Cultured granule cells grown in serum‐containing medium with a “low K+” concentration (10 mM) underwent apoptosis after maturation for 5 days in vitro (5 DIV), a time that coincides with the developmental decline in the activity of metabotropic glutamate receptors (mGluRs) coupled to polyphosphoinositide hydrolysis. The mGluR agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) prevented the development of low K+‐induced apoptosis and the presence of the drug was critical at 6 and 7 DIV, i.e., after the drop of mGluR activity. The neuroprotective action of 1S,3R‐ACPD was prevented by the mGluR antagonist (RS)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) and was mimicked by N‐methyl‐d‐aspartate or carbamylcholine but not by agonists of the mGluR subtypes negatively linked to adenylyl cyclase. In cultures treated either with Li+—which reduced polyphosphoinositide response to concentrations of glutamate (5 µM) that approximate those physiologically present in the incubation medium—or MCPG, the development of low K+‐induced apoptosis already occurred at 4 DIV. Thus, the activation of mGluRs coupled to polyphosphoinositide hydrolysis by endogenous glutamate could contribute to protect cultured granule cells against apoptosis during early stages of maturation.
Comparison of Postasphyxial Resuscitation with 100% and 21% Oxygen on Cortical Oxygen Pressure and Striatal Dopamine Metabolism in Newborn Piglets
Authors: Huang Chao‐Ching , Yonetani Masahiko , Lajevardi Nasser , Delivoria‐Papadopoulos Maria , Wilson David F. , Pastuszko Anna
Published: July 2010

Abstract: The present study tests the hypothesis that ventilation with 100% O2 during recovery from asphyxia leads to greater disturbance in brain function, as measured by dopamine metabolism, than does ventilation with 21% oxygen. This hypothesis was tested using mechanically ventilated, anesthetized newborn piglets as an animal model. Cortical oxygen pressure was measured by the oxygen‐dependent quenching of phosphorescence, striatal blood flow by laser Doppler, and the extracellular levels of dopamine and its metabolites by in vivo microdialysis. After establishment of a baseline, both the fraction of inspired oxygen (FiO2) and the ventilator rate were reduced in a stepwise fashion every 20 min over a 1‐h period. For the subsequent 2‐h recovery, the animals were randomized to breathing 21 or 100% oxygen. It was observed that during asphyxia cortical oxygen pressure decreased from 36 to 7 torr, extracellular dopamine increased 8,300%, and dihydroxyphenylacetic acid and homovanillic acid decreased by 65 and 60%, respectively, compared with controls. During reoxygenation after asphyxia, cortical oxygen pressure was significantly higher in the piglets ventilated with 100% oxygen than in those ventilated with 21% oxygen (19 vs. 11 torr). During the first hour of reoxygenation, extracellular dopamine levels decreased to ∼200% of control in the 21% oxygen group, whereas these levels were still much higher in the 100% oxygen group (∼500% of control). After ∼2 h of reoxygenation, there was a secondary increase in extracellular dopamine to ∼750 and ∼3,000% of baseline for the animals ventilated with 21 and 100%, respectively. It is concluded that although 100% FiO2 after asphyxia increases cortical oxygenation compared with 21% FiO2, it also results in poorer recovery in dopamine metabolism and higher secondary release of striatal dopamine. The resulting increased extracellular levels of dopamine may exacerbate posthypoxic cerebral injury.
Effect of Cytidine on Membrane Phospholipid Synthesis in Rat Striatal Slices
Authors: Savci Vahide , Wurtman Richard J.
Published: July 2010

Abstract: Using rat striatal slices, we examined the effect of cytidine on the conversion of [3H]choline to [3H]‐phosphatidylcholine ([3H]PC), and on net syntheses of PC, phosphatidylethanolamine (PE), and phosphatidylserine, when media did or did not also contain choline, ethanolamine, or serine. Incubation of striatal slices with cytidine (50–500 µM) caused dose‐dependent increases in intracellular cytidine and cytidine triphosphate (CTP) levels and in the rate of incorporation of [3H]choline into membrane [3H]PC. In pulse‐chase experiments, cytidine (200 µM) also increased significantly the conversion of [3H]choline to [3H]PC during the chase period. When slices were incubated with this concentration of cytidine for 1 h, small (7%) but significant elevations were observed in the absolute contents (nmol/mg of protein) of membrane PC and PE (p < 0.05), but not phosphatidylserine, the synthesis of which is independent of cytidine‐containing CTP. Concurrent exposure to cytidine (200 µM) and choline (10 µM) caused an additional significant increase (p < 0.05) in tissue PC levels beyond that produced by cytidine alone. Exposure to choline alone at a higher concentration (40 µM) increased the levels of all three membrane phospholipids (p < 0.01); the addition of cytidine, however, did not cause further increases. Concurrent exposure to cytidine (200 µM) and ethanolamine (20 µM) also caused significantly greater elevations (p < 0.05) in tissue PE levels than those caused by cytidine alone. In contrast, the addition of serine (500 µM) did not enhance cytidine's effects on any membrane phospholipid. Exposure to serine alone, however, like exposure to sufficient choline, increased levels of all three membrane phospholipids significantly (p < 0.01). These data show that exogenous cytidine, probably acting via CTP and the Kennedy cycle, can increase the synthesis and levels of membrane PC and PE in brain cells.
Characterization of Sialyltransferase‐IV Activity and Its Involvement in the c‐Pathway of Brain Ganglioside Metabolism
Authors: Freischütz Bettina , Saito Megumi , Rahmann Hinrich , Yu Robert K.
Published: July 2010

Abstract: To characterize the sialyltransferase‐IV activity in brain tissues, the activities of GM1b‐, GD1a‐, GT1b‐, and GQ1c‐synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1b, or a cod brain ganglioside mixture as the substrate. The GD1a‐synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg2+ or Mn2+ and Triton CF‐54 for its full activity. The Vmax value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent Km values for CMP‐sialic acid and GM1 were 172 and 78 µM, respectively. Cichlid fish and rat brains also contained GM1b‐, GT1b‐, and GQ1c‐synthase activities. The ratio of GM1b‐, GD1a‐, and GT1b‐synthase activities in fish brain was 1.00:0.89:1.13, respectively, and in rat brain 1.00:0.60:0.63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [3H]CMP‐sialic acid produced radiolabeled GQ1c. It is interesting that the adult rat brain also contains an appreciable level of GQ1c‐synthase activity despite its very low concentrations of c‐series gangliosides. The GD1a‐ or GQ1c‐synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltransferase‐IV reactions. Thus, the GD1a‐synthase activity was inhibited by GA1 and GD1b, but not by LacCer, GM3, or GD3. In a similar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3. The GD1a‐synthase activity directed toward endogenous GM1 was inhibited by GA1 or GT1b, whereas the endogenous GT1b‐synthase activity was suppressed by GA1 or GM1. GA1, GM1, and GD1b did not affect the endogenous GM3‐ and GD3‐synthase activities. These results clearly demonstrate that sialyltransferase‐IV in brain tissues catalyzes the reaction for GQ1c synthesis in the c‐pathway as well as the corresponding steps in the asialo‐, a‐, and b‐pathway in ganglioside biosynthesis.