bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Perinatal Vitamin B6 Deficiency on Dopaminergic Neurochemistry
Authors: Guilarte Tomas R. , Wagner Henry N. , Frost J. James
Published: October 2006

Abstract: Long‐Evans dams were fed either a vitamin B6‐deficient or a control diet from day 13–14 of gestation and throughout lactation. A control pair‐fed group was also included because of differences in food intake between vitamin B6‐deficient and control ad libitum dams. The progeny of vitamin B6‐deficient dams had all the classic symptoms of B6 deficiency. These included weight loss, ataxia, tremor, and epileptic seizures. Concentrations of the neurotransmit‐terdopamine (DA), and its metabolites 3,4‐dihydroxyphen‐ylacetic acid (DOPAC) and homovanillic acid (HVA), as well as D‐2 dopamine receptor binding, 3,4‐dihydroxy‐phenylalanine (DOPA) decarboxylase activity, and vitamin B6 levels were measured in the corpus striatum of progeny at 7, 14, and 18 days after birth. Striatal DA and HVA levels were significantly decreased in B6‐deficient animals when compared to ad libitum or pair‐fed controls. Daily injections of vitamin B6 to deprived animals from the 14th to 18th day after birth improved the abnormal movement and normalized the concentration of DA but not of HVA in corpus striatum. Striatal D‐2 dopamine receptor binding using [3H]spiperone as ligand was significantly reduced in 18‐day‐3old animals as compared to ad libitum and pair‐fed controls. No significant differences were found at 14 days. The administration of vitamin B6 to deprived animals did not raise the level of D‐2 receptor binding during the period of observation. Scatchard plots indicated that the differences in binding were due to changes in receptor number and not in KD. Corpus striatum DOPA decarboxylase activity with and without the addition of exogenous pyridoxal phosphate was significantly reduced in 14‐ and 18‐day‐old animals when compared to pair‐fed controls. Vitamin B6 levels in corpus striatum of deprived animals were significantly lower than control at all ages, substantiating the effect of the maternal deprivation. The results of these experiments suggest that perinatal vitamin B6 deficiency produces significant alterations in the development of the dopaminergic system in the corpus striatum of the neonatal rat.
Atrial Natriuretic Factors Stimulate Accumulation and Efflux of Cyclic GMP in C6–2B Rat Glioma and PC12 Rat Pheochromocytoma Cell Cultures
Authors: Fiscus Ronald R. , Robles Beemeth T. , Waldman Scott A. , Murad Ferid
Published: October 2006

Abstract: Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6–2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates paniculate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6–2B and PC12 cells with the rank order of potency being atriopeptin HI = atriopeptin II ≥ human atrial natriuretic polypeptide ≥ atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6–2B cells fourfold and in PC12 cells 12‐fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6–2B cells (47‐fold) and PC12 cells (12‐fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (250 μM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells. Although the function of cyclic GMP in neural tissue is at present unclear, the cyclic GMP response observed in C6–2B and PC12 cells may provide important clues concerning the molecular mechanisms of action of ANF in the central and peripheral nervous systems. Since a significant amount of the generated cyclic GMP is released from these cells in response to ANF, extracellular as well as intracellular cyclic GMP may be important in mediating the actions of ANF in neural tissues.
In Vivo Studies on the Extracellular, and Veratrine‐Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion
Authors: Butcher Steven P. , Hamberger Anders
Published: October 2006

Abstract: The effects of corticostriatal deafferentation (de‐cortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine‐releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra‐and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. γ‐Aminobutyric acid (GABA), tau‐nne (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month postlesion. The intracellular pool of Glu and Asp was also reduced in kainate‐lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of nonneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion‐induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood‐brain bamer transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine. GABA, Tau, and PEA effluxes were also decreased in kainate‐lesioned striata. These findings are consistent with the proposed roles of an acidic amino acid as the corticostriatal transmitter, and of GABA as a transmitter in intrinsic striatal neurons. The existence of releasable pools of Tau and PEA within kainate‐sensitive striatal neurons would also appear to be likely.
Cellular Origins of Endogenous Amino Acids Released into the Extracellular Fluid of the Rat Striatum During Severe Insulin‐Induced Hypoglycemia
Authors: Butcher Steven P. , Sandberg Mats , Hagberg Henrik , Hamberger Anders
Published: October 2006

Abstract: The effect of severe insulin‐induced hypoglycemia on the extracellular levels of endogenous amino acids in the rat striatum was examined using the brain microdialysis technique. A characteristic pattern of alterations consisting of a 9–12‐fold increase in aspartate (Asp), and more moderate increases in glutamate (Glu), taurine (Tau), and γ‐aminobutyric acid (GABA), was noted following cessation of electroencephalographic activity (isoelectricity). Glutamine (Gln) levels were reduced both during and after the isoelectric period and there was a delayed increase in extracellular phosphoethanolamine (PEA) content. The effects of decortication and excitotoxin lesions on the severe hypoglycemia‐evoked efflux of endogenous amino acids in the striatum were also examined. Decortication reduced the release of Glu and Asp both 1 week and 1 month post‐lesion. The efflux of other neuroactive amino acids was not affected significantly. In contrast, GABA, Tau, and PEA efflux was attenuated in kainate‐lesioned striata. Glu and Asp release was also reduced under these conditions, and a smaller decrease in extracellular Gln was noted. These data suggest that GABA, Glu, and Asp are released primarily from their transmitter pools during severe hypoglycemia. The releasable pools of Tau and PEA appear to be located in kainate‐sensitive striatal neurons. The significance of these results is discussed with regard to the excitotoxic theory of hypoglyce‐mic cell death.
Excitatory Amino Acid Receptor Potency and Subclass Specificity of Sulfur‐Containing Amino Acids
Authors: Pullan L. M. , Olney J. W. , Price M. T. , Compton R. P. , Hood W. F. , Michel J. , Monahan J. B.
Published: October 2006

Abstract: The sulfur‐containing amino acids, l‐and d‐cysteate, l‐cysteine, l‐and d‐cysteine sulfinate, l‐and d‐cysteine‐S‐sulfate, l‐cystine, l‐and d‐homocysteate, l‐and d‐homocysteine sulfinate, l‐homocysteine, l‐serine‐O‐sulfate, and taurine were tested in two excitatory amino acid receptor functional assays and in receptor binding assays designed to label specifically the AAl/N‐methyl‐d‐aspartate (NMDA), AA2/quisqualate, and AA3/kainate receptor recognition sites, as well as a CaCla‐dependent l‐2‐amino‐4‐phosphonobutanoate site, and a putative glutamate uptake site. Agonist efficacies were determined by chick retinal excitotoxicity and stimulated sodium efflux from rat brain slices. d‐Homocysteine sulfinate, l‐homocysteate, and l‐serine‐O‐sulfate had affinities most selective for the NMDA binding site, whereas the binding affinities of d‐cysteate, d‐cysteine sulfinate, d‐homocysteate, and l‐homocysteine sulfinate were less selective. However, the correlation of agonist activity sensitive to blockade by d‐2‐amino‐7‐phosphonoheptanoate or d‐2‐amino‐5‐phosphonopentanoate in the functional assays with affinity in the NMDA binding assay (r= 0.87, p < 0.005 and r= 0.98, p < 0.005 for excitotoxicity and sodium efflux, respectively) allows characterization of these sulfur‐containing amino acids as acting at NMDA subclass receptors. l‐Homocysteate, which has been found in the brain, and l‐serine‐O‐sulfate are selective agonists and could serve as endogenous neurotransmitters at the NMDA receptor.
Antibodies to Cholinergic Neurons in Alzheimer's Disease
Authors: Chapman Joab , Bachar Orit , Korczyn Amos D. , Wertman Eliyahu , Michaelson Daniel M.
Published: October 2006

Abstract: Alzheimer's disease (AD) is associated with degenerative changes in nuclei of the basal forebrain which provide most of the cholinergic input to the cortex and hippocampus and with a reduction in presynaptic cholinergic parameters in these areas. Although the etiology and pathogenesis of AD are not known, several reports indicate the involvement of immunological mechanisms. In the present work we examined the existence of antibodies in sera of AD patients that bind specifically to cholinergic neurons. As antigens we employed the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogeneous and cross‐react antigenically with human and other mammalian cholinergic neurons. Our findings show that immunoglobulins from sera of AD patients bind to a specific antigen (molecular mass 200 kilo‐daltons) in the cell bodies and axons of Torpedo electromotor neurons and that the levels of such antibodies are significantly higher in AD patients than in controls. The possible role of these antibodies in the cholinergic dysfunction in AD and their diagnostic potential are discussed.
Characterization of Bradykinin‐Induced Phosphoinositide Turnover in Neurohybrid NCB‐20 Cells
Authors: Chuang De‐Maw , Dillon‐Carter Ora
Published: October 2006

Abstract: Phosphoinositide hydrolysis was studied in neurohybrid NCB‐20 cells prelabeled with myo‐[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IPO in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 μM. The bradykinin response was robust (10‐fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D‐Arg‐(Hyp3 Thi5,8, D‐Phe7)‐bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine Hi receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin‐induced [3H]IPi accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carba‐chol‐induced accumulation in the same cell line. In contrast, using related neurohybrid NG108‐15 cells, brady‐kinin‐induced [3H)IPi accumulation was found to require much less lithium. In the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]‐inositol bis‐ and trisphosphate. Basal and bradykinin‐in‐duced phosphoinositide breakdown was inhibited by 4β‐phorbol 12, 13‐dibutyrate, but was unaffected by the biologically inactive 4β‐phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the brady‐kinin‐induced [3H]IP1 accumulation, without affecting basal activity. These data might suggest that more than one type of GTP binding protein is involved in the accumulation of IP1. Preincubation of bradykinin (400 nM) with cells resulted in a time‐dependent loss of the ability of bradykinin to stimulate phosphoinositide hydrolysis; more than 50% of the activity was lost after 45 min exposure at 37°C. The bradykinin response was markedly attenuated by prestimulation with bradykinin; this desensitization was time‐dependent with a maximal effect observed after about 1 h prestimulation. Thus, the robust response of bradykinin on phosphoinositide turnover in NCB‐20 cells may serve as another interesting model in the study of its role in some bradykinin‐mediated physiological events.
Melatonin Binding Sites
Authors: Vaněček Jiří
Published: October 2006

Abstract: The distribution and characterization of specific melatonin binding sites were studied using 125I‐melatonin. Autoradiography revealed only three sites of specific melatonin binding in brain: the suprachiasmatic nuclei, the median eminence, and the small part of choroid plexus at the caudal end of the fourth ventricle. Two other sites were detected outside the CNS: the anterior pituitary and the retina. The specific binding of 125I‐melatonin was saturable and reversible. The dissociation constant (KD) of the binding sites was 60 pM. The concentration of the binding sites (Bmax) in the median eminence was 26 fmol/mg protein, and in the pituitary 3 fmol/mg protein. Specificity of the binding sites was tested by displacement of 125I‐melatonin. The order of potency—melatonin ≪N‐acetyl‐5‐hydroxytryptamine < 5‐methoxytryptamine ≪ 5‐hydroxytryptamine = 3,4‐dihydroxyphenylethylamine = noradrenaline—shows high specificity of the binding sites for melatonin.
Stimulation of Distinct D2 Dopaminergic and α2‐Adrenergic Receptors Induces Light‐Adaptive Pigment Dispersion in Teleost Retinal Pigment Epithelium
Authors: Dearry Allen , Burnside Beth
Published: October 2006

Abstract: In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4‐dihydroxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half‐maximal effect at approximately 2 nM; clonidine, at 1 μM. Dopamine‐induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by Dl or α‐adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a Dl dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine‐induced dispersion was inhibited by the α2‐adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and α2‐adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark‐adaptive pigment aggregation. Together, our results suggest that stimulation of D2 dopaminergic or α2‐adrenergic receptors on teleost RPE induces light‐adaptive pigment granule dispersion at least in part by inhibiting adenylate cyclase activity in these cells. We suggest that modulation of RPE cAMP levels by catecholamines supplied from either the retina or the choroid could contribute to the regulation of RPE responses to both light and circadian signals.
Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area: Effects of Adenosine and Baclofen
Authors: Burke Stephan P. , Nadler J. Victor
Published: October 2006

Abstract: Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3‐derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA 1 area were employed to test the effects of adenosine‐and 7‐aminobutyrate (GABA)‐related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+‐dependent and originated predominantly from the CA3‐derived pathways. Adenosine reduced the K+‐evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 μM 8‐phenyltheophylline. The order of potency for adenosine analogues was as follows: l‐N6‐phenylisopropyl‐adenosine > N6‐cyclohexyladenosine > d‐N6‐phenylisopro‐pyladenosine ≅ 2‐chloroadenosine > adenosine ≫5′‐N‐ethylcarboxamidoadenosine. 8‐Phenyltheophylline (10 μM) by itself enhanced glutamate/aspartate release, whereas di‐pyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer col‐lateral‐commissural‐ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, l‐N6‐phenylisopropyladenosine, nor 8‐phenyltheophylline affected the release of glutamate or aspartate evoked by 10 μM ve‐ratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage‐sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage‐sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.
Brain Slice Glucose Utilization
Authors: Newman George C. , Hospod Frank E. , Patlak Clifford S.
Published: October 2006

Abstract: The metabolism of 2‐deoxyglucose has been studied in 540 μm and 1,000 μm hypothalamic brain slices. Slice 2‐deoxyglucose (2DG) and 2‐deoxyglucose‐6‐phosphate (2DG6P) levels were measured after tissue homogenization and perchloric acid extraction. By analyzing the uptake and washout kinetics with nonlinear least‐squares methods, we have determined the rate constants for three‐, four‐, or five‐parameter kinetic models and obtained a value for the in vitro lumped constant (LC). The kinetic analysis reveals a small, slowly decaying, 2DG component that is not predicted by any of the models. If this component is treated as a separate, parallel compartment, then the four‐ and five‐parameter models are essentially equivalent. To compare our data to prior in vivo data, we combined 2DG and 2DG6P to produce Ci*, the total slice radioactivity, and analyzed the first 45 min of uptake. These data were fit best by a three‐parameter model and the slowly decaying pool was not identified. Calculation of glucose utilization from total tissue radioactivity, measured by whole slice homogenization and by image analysis of au‐toradiograms, showed excellent correlation between the two methods. Image analysis of radioactivity in the suprachias‐matic nucleus, which is present in these slices, revealed a spontaneous diurnal variation in in vitro glucose utilization in close quantitative agreement with prior in vivo measurements. The kinetic analysis of the 1,000 μm slice was qualitatively similar to that of the 540 μm slice but revealed an increase in the LC and a large decrease in k1 as well as the expected large increase in the hexokinase rate constant, k3. Overall, in vitro glucose utilization increased by about 60%. These results are consistent with our prior studies of the 1,000 μm slice and support our interpretation that the 1,000 μm slice is an excellent in vitro model for brain ischemia without infarction.
Characterization of In Vivo Dopamine Release as Determined by Brain Microdialysis After Acute and Subchronic Implantations: Methodological Aspects
Authors: Westerink B. H. C. , De Vries J. B.
Published: October 2006

Abstract: Infusion of tetrodotoxin (TTX) through the dialysis membrane and perfusion with calcium‐free Ringer solution (calcium depletion) were used to evaluate the dopamine release determined by in vivo brain dialysis. Several hours after implantation, the dopamine release recorded by the U‐shaped cannula did not respond to calcium depletion and was only partly (˜50%) TTX dependent. The half‐life of the TTX‐independent dopamine overflow was determined to be 2 h. In contrast, when a transstriatal cannula was used, the dopamine output displayed calcium and TTX dependency. Differences in the dimensions of the two types of probes are a likely explanation for the observed effects. Twenty‐four hours after implantation, both types of cannula fulfilled the criteria of calcium and TTX dependency. The results indicate that infusion of TTX‐contain‐ing or calcium‐free Ringer solution can be used to estimate the functional damage caused by the implantation of the cannula.
Brain Indoleamines in Alloxan‐ and Streptozotocin‐Induced Diabetic Rats
Authors: Mello Luiz E. A. M. , Bortolotto Zuner A. , Cavalheiro Esper A.
Published: October 2006

Abstract: Previous work by other authors has shown that alloxan‐induced diabetes increases whereas streptozotocin‐induced diabetes does not alter nonesterified fatty acid (NEFA) plasma levels. The present study replicates these results and demonstrates that fasted, streptozotocin‐in‐duced diabetic animals also have increased NEFA levels. In addition, brain levels of 5‐hydroxytryptamine (5‐HT) and of its immediate precursor and metabolite were measured. Alloxan‐ and fasted, streptozotocin‐induced diabetic rats showed significant increases in brain indoleamine concentrations, whereas fed, streptozotocin‐induced diabetic rats had unchanged levels of the same compounds. Levels of brain indoleamines exhibited a strong positive correlation with wet‐dog shakes (an index of 5‐HT activity) elicited by hippocampal stimulation. Blockade of wet‐dog shakes by 5‐HT receptor antagonists strengthens the proposal that this behavior is a good index of central 5‐HT activity. The increased content of brain indoleamines in alloxan‐ and fasted, streptozotocin‐induced diabetic rats may be related to the increased NEFA plasma levels seen in the same animals. This hypothesis is supported by the positive correlation demonstrated between NEFA and 5‐HT levels. In conclusion, it is suggested that alloxan‐induced diabetes may represent a useful model for studying the various behavioral changes known to occur in diabetics.
Cooperative Modulation of [3H]MK‐801 Binding to the N‐Methyl‐d‐Aspartate Receptor‐Ion Channel Complex by l‐Glutamate, Glycine, and Polyamines
Authors: Ransom Richard W. , Stec Nancy L.
Published: October 2006

Abstract: In extensively washed rat cortical membranes [3 H](+)‐ 5 ‐ methyl ‐ 10,11 ‐ dihydro ‐ 5 H‐ dibenzo [a,d]cyclohepten‐5,10‐imine ([3H]MK‐801) labeled a homogeneous set of sites (Bmax=1.86 pmol/mg protein) with relatively low affinity (KD= 45 nM). l‐Glutamate, glycine. and spermidine produced concentration‐dependent increases in specific [3H]MK‐801 binding due to a reduction in the Kd of the radioligand. In the presence of high concentrations of l‐glutamate, glycine, or spermidine, the KD values for [3H]MK‐801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK‐801 binding affinity as follows: l‐glutamate + glycine, KD= 6.2 nM; l‐glutamate + spermidine, KD= 2.2 nM:
D1 and D2 Dopamine Receptors in Caudate‐Putamen of Nonhuman Primates (Macaca fascicularis)
Authors: Madras Bertha K. , Fahey Michele A. , Canfield Don R. , Spealman Roger D.
Published: October 2006

Abstract: D1 and D2 dopamine receptors were characterized in the caudate‐putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]‐spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high‐affinity binding site (KD, 0.352 ± 0.027 nM) with a density (Bmax) of 35.7 ± 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 ± 0.007 nMand the density was 25.7 ± 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS‐SKF 38393 was the most selective D1 agonist, whereas (+)‐4‐propyl‐9‐hydroxynaphthoxazine [(+)‐PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R‐SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM‐09151–2 was the most selective for the D2 site. cis‐Flupentixol and (S)‐butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/ SCH 23388, R‐SKF 83692/RS‐SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)‐sulpiride and (+)‐PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.
Kinetics of Choline Uptake into Isolated Rat Forebrain Microvessels: Evidence of Endocrine Modulation
Authors: Shimon Mazal , Egozi Yakov , Kloog Yoel , Sokolovsky Mordechai , Cohen Sasson
Published: October 2006

Abstract: The active uptake of [methyl‐3H]choline into isolated rat brain microvessel suspension was studied as a likely guide to the transport of choline across the blood‐brain barrier. The method consisted primarily of incubation of the suspension with a fixed concentration of labeled choline in the presence of increasing concentrations of un‐labeled choline or any other inhibitor (I) of active uptake, denned as the difference in uptake at 37° and 0°C. From the linear regression of (1/V) against [I], the following values of Kmax (nmol g−1 min−1) and Km (μM) were obtained for choline: 2‐month‐old males, 10.6 ± 3.8 and 6.1 ± 0.9; 3‐month old random females, 28.4 ± 5.9 and 12.6 ± 4.0; females at metaestrus, 17.8 ± 10.3 and 8.3 ± 5.0; at diestrus, 31.1 ± 9.3 and 13.0 ± 2.6; at proestrus, 54.9 ± 2.2 and 14.0 ± 1.5; at estrus, 19.2 ± 2.2 and 2.6 ± 1.7. The differences between males and random females (p < 0.018) and between females at proestrus and estrus (p < 0.005) are significant. It is suggested that these inter‐ and intrasex variations in choline uptake reflect a dynamic adjustment of supply in accordance with brain demand for choline at the time of assay. Hemicholinium‐3 was an effective inhibitor of choline uptake, Ki= 14.0 ± 8.5 μM; dimethylaminoethanol was much less effective; and imipramine had no measurable effect.
Presence of Kynurenic Acid in the Mammalian Brain
Authors: Moroni F. , Russi P. , Lombardi G. , Beni M. , Carlà V.
Published: October 2006

Kynurenic acid, a tryptophan metabolite able to antagonize the actions of the excitatory amino acids, has been identified and measured for the first time in the brain of mice, rats, guinea pigs, and humans by using an HPLC method. Its content was 5.8 ± 0.9 in mouse brain, 17.8 ± 2.0 in rat brain, 16.2 ± 1.5 in guinea pig brain, 26.8 ± 2.9 in rabbit brain, and 150 ± 30 in human cortex (pmol/g wet wt, mean ± SE). The regional distribution of this molecule was uneven. In rats, guinea pigs, and rabbits, the brainstem was the area richest in this compound. Tryptophan administration (100–300 mg/kg, i.p.) to rats resulted in a significant increase of the brain content of kynurenic acid. Similarly, 1 h after probenecid administration (200 mg/kg, i.p.), the brain content of kynurenate increased by fourfold, thus suggesting that its turnover rate is relatively fast.
Pharmacological Characterization of Rapidly Accumulated Adenosine by Dissociated Brain Cells from Adult Rat
Authors: Geiger J. D. , Johnston M. E. , Yago V.
Published: October 2006

Mechanically dissociated brain cells from adult rats were used to study biochemically and pharmacologically their capacity to accumulate rapidly [3H]adenosine. The assay, which used an inhibitor‐stop method to prevent further uptake into cells, was characterized with respect to protein and optimal substrate concentrations, and incubation times that ranged from 5 to 180 s. The accumulation of [3H]adenosine using 15‐s incubation periods, conditions under which < 10% of accumulated [3H]adenosine was metabolized, was best described kinetically by a two‐component system with Km and Vmax values for the high‐affinity component of 0.8 μM and 6.2 pmol/mg protein/15 s and for the low‐affinity component 259 μM and 2,217 pmol/mg protein/15 s, respectively. The potencies with which nucleosides, adenosine deaminase resistant adenosine receptor agonists, and nucleoside uptake inhibitors competed for these uptake components were determined. Of the nucleosides examined, adenosine was the „preferred” substrate for the uptake site. The Ki value of adenosine for the high‐affinity component was 10.7 μM. Inosine and uridine competed for a single lower affinity uptake system: Ki values were 142 and 696 μM, respectively. Nucleoside uptake inhibitors—nitrobenzylthioinosine, dipyridamole. and dilazep—were the most potent inhibitors of [3H]adenosine accumulation tested: the Ki values for the high‐affinity system were 0.11, 1.3, and 570 nM, respectively. The adenosine analogs S‐phenylisopropyladenosine, R‐phenylisopropyladenosine, and cyclohexyladenosine inhibited the high‐affinity component with Ki values of 2.3, 9.3, and 14.5 μM, respectively. N‐Ethylcarboxamidoadenosine competed for a single lower affinity uptake system; Ki, 292 μM. These results indicate that dissociated brain cells avidly take up [3H]adenosine and that the pharmacological profile for nucleoside uptake is similar to that for [3H]nitrobenzylthioinosine binding to putative nucleoside transport sites when uptake studies are conducted using brief incubation intervals.
Acetylcholinesterases from Musca domestica and Drosophila melanogaster Brain Are Linked to Membranes by a Glycophospholipid Anchor Sensitive to an Endogenous Phospholipase
Authors: Fournier Didier , Bergé Jean‐Baptiste , de Almeida Maria‐Lucia Cardoso , Bordier Clément
Published: October 2006

Abstract: The sensitivity of acetylcholinesterases (AChEs) from Musca domestica and from Drosophila melanogaster to the phosphatidylinositol‐specific phospholipase C from Bacillus cereus and to the glycosylphosphatidylinositol‐specific phospholipase C from Trypanosoma brucei was investigated. B. cereus phospholipase C solubilizes membrane‐bound AChE, and both phospholipases convert amphiphilic AChEs into hydrophilic forms of the enzyme. The Upases uncover an immunological determinant that is found on other glycosylphosphatidylinositol‐anchored membrane proteins after the same treatment. This immunological determinant is also present on the native hydrophilic form of AChE. The polypeptide bearing the active site of the membrane‐bound enzyme migrates faster during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis than the same polypeptide from the soluble enzyme. We conclude that AChE from insect brain is attached to membranes via a glycophospholipid anchor. This anchor is covalently linked to the polypeptide bearing the active esterase site of the enzyme and can be cleaved by an endogenous lipase.
Uptake of γ‐Aminobutyric Acid by a Synaptic Vesicle Fraction Isolated from Rat Brain
Authors: Fykse Else M. , Fonnum Frode
Published: October 2006

Abstract: γ‐Aminobutyric acid (GABA) was taken up by a MgATP‐dependent mechanism into synaptic vesicles isolated by hypoosmotic shock and density gradient centrifugation. The properties of the vesicular uptake differed clearly from those of synaptosomal and glial uptake, both with respect to Na+, Mg2+, and ATP dependence and with respect to response to general GABA uptake inhibitors such as nipecotic acid, diaminobutyric acid, and β‐alanine. The uptake showed a Km of 5.6 mM and a net uptake rate of 1,500 pmol/min/mg of protein. It is suggested that the vesicular uptake of GABA is driven by an electrochemical proton gradient generated by a Mg2+‐ATPase.
Presence and Measurement of Imidazoleacetic Acid, a γ‐Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid
Authors: Khandelwal Jai K. , Prell George D. , Morrishow Albert Marcus , Green Jack Peter
Published: October 2006

Abstract Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic‐mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N,15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n‐butyl(N‐ethoxycarbonyl)imidazole acetate and n‐butyl(N‐methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean ± SEM) of free IAA were 140 ± 14 pmol/g and 2.7 ± 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at γ‐aminobutyrate receptors, it merits examination as a regulator in brain.
Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood‐Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries
Authors: Koenig Harold , Goldstone Alfred D. , Lu Chung Y. , Trout Jerome J.
Published: October 2006

Abstract We recently presented evidence that the reversible opening of the blood‐brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15‐fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (<15 s), concentration‐ and time‐dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, α‐ difluoromethylornithine (DFMO, 10 m M). Mannitol (1 M), as well as 2 M urea, evoked a two‐ to fivefold increase in the temperature‐sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2‐deoxy‐D‐[1‐3H]glucose (DG), but not α‐[1‐14C]aminoisobutyrate, during a 2‐min incubation. DFMO (10 mM) abolished 1 M mannitol‐mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)‐induced stimulation of ODC activity and membrane transport processes was Ca2+‐dependent and verapamil‐ and nisoldipine‐sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two‐ to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC‐controlled polyamine synthesis. The effects of 10 nM PMA and 1 M mannitol were additive, suggesting that hyperosmolal stimulation of ODC‐activated polyamine synthesis does not involve protein kinase C. These data support the hypothesis that ODC‐activated polyamine synthesis and Ca2+ influx (via Ca2+ channels) play a key role in mediating the effects of hyperosmolality on BBB permeability.
Modulation of Dendritic Release of Dopamine by N‐Methyl‐D‐Aspartate Receptors in Rat Substantia Nigra
Authors: Araneda Ricardo , Bustos Gonzalo
Published: October 2006

Abstract: A superfusion system was used to study the effects of excitatory amino acids (EAA) on release of [3H]dopamine ([3H]DA) previously taken up by rat substantia nigra (SN) slices. The EAA tested (20–250 μM), with the exception of quisqualate and kainate, markedly evoked [3H]DA release from nigral slices when Mg2+ ions were omitted from the superfusion medium. The EAA receptor agonists exhibited the following relative potency in stimulating [3H]DA release: l‐glutamate (L‐Glu) > N‐methyl‐d‐aspartate (NMDA) > NM(D,L)A > d‐Glu ≫ quisqualate = kainate. d‐2‐Amino‐5‐phosphonovalerate (100–200 μ.M), an antagonist for NMDA receptors, substantially reduced [3H]DA release evoked by l‐Glu or NMDA. In contrast, l‐Glu diethyl ester (100–200 μM) produced a lesser blocking effect on [3H]DA release evoked by the EAA. Further experiments showed that the NMDA‐mediated release of [3H]DA was totally suppressed by the omission of Ca2+ or by the addition of tetrodotoxin (0.1 μM) to the superfusion medium. In addition, strychnine, an antagonist for glycine (Gly) receptors, significantly decreased NMDA (100 μM)‐evoked as well as glycine (100 μM‐evoked release of [3H]DA from nigral slices. The results shown support the idea that activation of NMDA subtype receptors in SN may trigger a Ca2+‐dependent release of DA from dendrites of nigro‐striatal DA‐containing neurons. Furthermore, a transsynaptic mechanism that may partially involve Gly‐containing interneurons is proposed to account for some of the events mediating NMDA receptor activation and DA release in SN.
Nonneuronal Localization for Steroid Converting Enzyme: 3α‐Hydroxysteroid Oxidoreductase in Olfactory Tubercle of Rat Brain
Authors: Krieger Neil R. , Scott Robert G.
Published: October 2006

Abstract: 3α‐Hydroxysteroid oxidoreductase (EC 1.1.1.50) was localized in the rat brain by cryostat sectioning, microassay, and neurochemical lesions. Single 16‐μm sections were cut, homogenized, and assayed. In the olfactory tubercle 3α‐hydroxysteroid oxidoreductase activity is high in the piaglial layer at the surface, 20‐fold lower at a depth of 50 μm, and 50‐fold lower at a depth of 200 μm. A similar pattern of activity was seen in the olfactory bulb, the interpeduncular nucleus, the frontal pole of the cortex, and the frontoparietal cortex. When kainic acid, a toxin that destroys neurons but leaves glia and axons of passage intact, was injected into the olfactory tubercle, 3α‐hydroxysteroid oxidoreductase activity was undiminished whereas glutamic acid decarboxylase activity was reduced by 80%. This laminar distribution and insensitivity to kainic acid are consistent with a nonneuronal localization. The high concentration of astrocytes in the pia–glial layer, where 3α‐hydroxysteroid oxidoreductase activity is highest, leads us to suggest that this enzyme is localized to astrocytes. The presence of particular enzymes in some brain regions and not in others determines which products are synthesized and which are inactivated in those regions. Thus, the location of 3α‐hydroxysteroid oxidoreductase and other steroid converting enzymes can affect the activity of neuronal circuits and the behaviors regulated by those circuits.
Direct Evidence That Excitotoxicity in Cultured Neurons Is Mediated via N‐Methyl‐D‐Aspartate (NMDA) as well as Non‐NMDA Receptors
Authors: Frandsen Aase , Drejer Jørgen , Schousboe Arne
Published: October 2006

Abstract: Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L‐glutamate, kainate (KA), N‐methyl‐D‐aspartate (NMDA), or RS‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L‐aspartic acid β‐hydroxamate was added at 500 μM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate‐induced cytotoxicity by blocking only one of the glutamate receptor subtpes with either the NMDA receptor antagonist D‐(‐)‐2‐amino‐5‐phosphonopentanoate (APV) or with one of the specific non‐NMDA antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non‐NMDA glutamate agonists with no effect on NMDA‐induced cell death. Likewise, APV prevented NMDA‐induced cell death without affecting the KA‐ or AMPA‐induced neurotoxicity. It is concluded that EAA‐dependent neurotoxicity is induced by NMDA as well as non‐NMDA receptors.
Effect of Age on K+‐Induced Cytosolic Ca2+ Changes in Rat Cortical Synaptosomes
Authors: Ciovannelli Lisa , Pepeu Giancarlo
Published: October 2006

Abstract: 45Ca2+ uptake and cytosolic Ca2+ Concentrations ([Ca2+]i) were measured in synaptosomes prepared from the cerebral cortex of 3‐, 16‐, and 24‐month‐old male Charles River Wistar rats. Electron‐microscopic examination demonstrated no morphological differences between the synaptosomes prepared from 3‐ and 24‐month‐old rats. The fast phase of Ca2+ uptake was reduced in the 24‐month‐old animals as compared to the 3‐month‐old ones (‐23%. p < 0.001), whereas no difference was found between the 16‐and the 3‐month‐old rats. Age did not modify [Ca2+]i, as measured by the quin 2 technique, both at rest and immediately after depolarization with 50 mM K+. The Ca2+ load following depolarization was cleared in about 13 min in the 3‐month‐old rats. The rate of clearance was significantly slower both in the 16‐ (p <0.01) and in the 24‐month‐old rats (p < 0.0001). The addition of verapamil (60 μM) after depolarization restored [Ca2+]i to resting level in aged rats at the same rate as in young rats. A prolonged Ca2+ influx, therefore, may be responsible for the slower clearance of Ca2+load in aged rats.
Binding of Crotoxin, a Presynaptic Phospholipase A2Neurotoxin, to Negatively Charged Phospholipid Vesicles
Authors: Radvanyi Frangois , Saliou Bernard , Lembezat Marie‐Pierre , Bon Cassian
Published: October 2006

Abstract: Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of < 1 μM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to <1 μM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.
ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes
Authors: Terrian D. M. , Hernandez P. G. , Rea M. A. , Peters R. I.
Published: October 2006

Abstract: Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin‐derived peptides and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+‐dependent manner by K+‐in‐duced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2‐chloro‐adenosine, inhibited the K+‐stimulated release of both dynorphin B and dynorphin A(l‐8). 2‐Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D‐aspartic acid to deplete the cytosolic, Ca2+‐independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+‐dependent pool of glutamic acid was significantly inhibited by NiCl2, 2‐chloroadenosine, 5′‐N‐ethylcarboxami‐doadenosine, cyclohexyladenosine, and R(‐)‐N6(2‐phenyl‐isopropyl)adenosine, but not by ATP. 2‐Chloroadenosine‐induced inhibition was reversed when the external CaCl2concentration was raised from 1.8 mMto 6 mM. 8‐Phen‐yltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2‐chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+‐evoked release of both glutamic acid and dynorphin A(l‐8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin‐derived peptides