bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Kainate Toxicity to Cerebellar Neurons In Vitro Is Analogous to Reperfusion Tissue Injury
Authors: Dykens James A. , Stern Arnold , Trenkner Ekkhart
Published: October 2006

The neuroexcitotoxin kainate has been used as a selective lesioning agent to model the etiology of a number of neurodegenerative disorders. Although excitotoxins cause susceptible neurons to undergo prolonged or repeated depolarization, the proximate metabolic pathology responsible for neuronal necrosis has remained elusive. We report here that kainate‐induced death of cerebellar neurons in culture is prevented by inhibiting the enzyme xanthine ox‐idase, a cellular source of cytotoxic superoxide radicals (O2‐). Moreover, neurons are also protected from excitotoxin‐induced death by the addition to the culture medium of either superoxide dismutase or mannitol, which scavenge superoxide and hydroxyl radicals, respectively, or serine protease inhibitor, which forestalls formation of xanthine oxidase. These findings indicate that excitotoxin‐induced neuronal degeneration is mediated by superoxide radicals generated by xanthine oxidase, a mechanism partially analogous to that proposed for tissue damage seen upon reperfusion of ischemic tissues.
Stress‐Related Changes in Cerebral Catecholamine and Indoleamine Metabolism: Lack of Effect of Adrenalectomy and Corticosterone
Authors: Dunn Adrian J.
Published: October 2006

Abstract: The concentrations of catecholamine and indoleamine metabolites were measured in intact and adrenal‐ectomized mice to determine whether adrenal hormones mediate or modulate the stress‐induced responses. Thirty minutes of footshock resulted in significant increases of the ratios of the dopamine (DA) catabolite, dihydroxyphenyl‐acetic acid (DOPAC), to DA in prefrontal cortex, nucleus accumbens, striatum, hypothalamus, and brainstem, and of homovanillic (HVA)/DA ratios in nucleus accumbens, striatum, amygdala, and hypothalamus. Ratios of 3‐meth‐oxy‐4‐hydroxyphenylethyleneglycol to norepinephrine (NE) were also increased in prefrontal cortex, nucleus accumbens, septum, amygdala, hypothalamus, hippocampus, and brainstem. The concentration of NE was decreased in amygdala. 5‐Hydroxyindoleacetic acid (5‐HIAA)/5‐hydroxytryptamine (5‐HT, serotonin) ratios and free tryptophan were also increased in every brain region. Very similar data were obtained from mice restrained for 30 min. Adrenalectomy resulted in increased HVA/DA ratios in prefrontal cortex and striatum, and 5‐HIAA/5‐HT in septum. The stress‐related changes were largely similar in adrenalectomized mice. Significant interactions between adrenalectomy and footshock treatment occurred in prefrontal cortical DOPAC/DA and hypothalamic NE which was depleted only in adrenalectomized mice, suggesting tendencies for these measures to be more responsive in adrenalectomized mice. Corticosterone administration (0.5–2.0 mg/kg s.c.) which resulted in plasma concentrations in the physiological range did not alter the concentrations of the cerebral metabolites measured in any region. We conclude that adrenal hormones do not mediate cerebral catecholamine or indoleamine metabolism in stress, although adrenalectomy may affect HVA and 5‐HIAA metabolism, and there was a tendency for catecholamines to be more sensitive to stress in adrenalectomized animals.
Is Inositol Bisphosphate the Product of A23187 and Carbachol‐Mediated Polyphosphoinositide Breakdown in Synaptosomes?
Authors: Brammer Michael J. , Hajimohammadreza Iradj , Sardiwal Sudesh , Weaver Keith
Published: October 2006

Abstract: Synaptosomes have been isolated from rat cerebral cortex and labelled in vitro with [32P]orthophosphate and myo‐[2‐3H]inositol. Subsequent addition of the Ca2+ ionophore A23187 in the presence of 2 mM extrasynaptosomal Ca2+ raised intrasynaptosomal free [Ca2+] to >2 μM from a resting level of 200 nMand led to rapid breakdown of polyphosphoinositides. This was accompanied by a small increase in the level of inositol monophosphate, greatly enhanced accumulation of inositol bisphosphate, but no detectable increase in inositol trisphosphate. Depolarising (25 mM) extrasynaptosomal K+ produced a smaller increase in intrasynaptosomal free [Ca2+] (to around 400 nM) and a proportional increase in inositol bisphosphate radioactivity. Carbachol (1 mM) alone elicited only limited polyphosphoinositide breakdown and inositol mono‐ and bisphosphate formation, but this was greatly increased in the presence of 25 mM K+. The effect of carbachol in the presence of depolarising K+ was time‐ and dose‐dependent and was antagonised by atropine (10 μM). There was no detectable accumulation of inositol trisphosphate in the presence of carbachol, K+, or carbachol plus K+, even after short (30 s.) incubations. The lack of inositol trisphosphate accumulation does not appear to result from rapid formation of inositol tetrakisphosphate or from enhanced breakdown of the trisphosphate in synaptosomes.
Chloride‐Dependent Uncoupling of Oxidative Phosphorylation by Triethyllead and Triethyltin Increases Cytosolic Free Calcium in Guinea Pig Cerebral Cortical Synaptosomes
Authors: Kauppinen Risto A. , Komulainen Hannu , Taipale Hannu T.
Published: October 2006

Abstract: Metabolically competent isolated cerebral cortical nerve terminals were used to determine the effects of triethyllead (TEL) and triethyltin (TET) on cytosolic free calcium ([Ca2+]c), on plasma and mitochondrial membrane potentials, and on oxidative metabolism. In the presence of physiological concentrations of extracellular ions, 20 μM TEL and 20 μM TET increase [Ca2+]c from 185 nM to 390 and 340 nM, respectively. A simultaneous depolarization of plasma membrane potential (ΔΨp) by only 3–4 mV occurs, a drop which is insufficient to open the voltage‐sensitive Ca2+ channels. In contrast, an instant and substantial depolarization of mitochondrial membrane potential (ΔΨp) upon addition of TEL and TET is evident, as monitored with saf‐ranine O fluorescence. At the same concentration, TEL and TET stimulate basal respiration of synaptosomes by 45%, induce oxidation of endogenous NAD(P)H, and reduce the terminal ATP/ADP ratio by 45%. Thus, TEL and TET inhibit ATP production of intrasynaptosomal mitochondria by a mechanism consistent with uncoupling of oxidative phosphorylation. This bioenergetic effect by TEL and TET can be prevented by omitting external chloride, and a concomitant reduction of the increase in [Ca2+]c by about 60% is observed. Uncoupling of mitochondrial ATP synthesis from oxidation, a process that is dependent on external chloride, is the main mechanism by which TEL and TET increase [Ca2+]c.
Altered Expression of the D1.1 Ganglioside in the Cerebellum of the Weaver Mouse
Authors: Johnstone Stephen R. , Stallcup William B.
Published: October 2006

Abstract: Expression of the D1.1 ganglioside was studied immuno‐histochemically in developing cerebella from normal and weaver mutant mice. In the normal cerebellum at postnatal day 7 (P7), D1.1 expression was restricted to the external granule‐cell layer (EGL). At later ages, D1.1 disappeared as the developing granule neurons ceased mitosis and began migrating toward the internal granule‐cell layer. In the weaver cerebellum, D1.1 was expressed in the EGL in apparently normal fashion at P7, but failed to disappear at later ages. As late as P35, D1.1 immunoreactivity was observed throughout the weaver cerebellar cortex. The relative amounts of D1.1 ganglioside in weaver and normal cerebella were compared by thin layer chromatography of total gangliosides, followed by overlay of the chromatogram with anti‐D1.1 and 125I‐labelled second antibody. Autoradiograms showed that at P12 and P35 the weaver tissue contains six‐ to tenfold more D1.1 than normal tissue. These findings suggest that one result of the weaver mutation is prolonged expression of D1.1. We speculate that the D1.1 ganglioside might be involved in adhesive interactions that regulate the timing of granule‐cell migration from the EGL. The prolonged expression of D1.1 could be responsible, in part, for the failure of granule‐cell migration in the weaver cerebellum.
Release of N‐Acetylaspartylglutamate on Depolarization of Rat Brain Slices
Authors: Zollinger Markus , Amsler Ursula , Do Kim Quang , Streit Peter , Cuénod Michel
Published: October 2006

Abstract: In a great number of investigations, evidence in favor of a neurotransmitter role of the N‐terminal‐blocked, acidic dipeptide N‐acetylaspartylglutamate (NAAG) has been accumulating. In fact, in some systems of the mammalian brain, almost all of the classical criteria for neurotransmitters have been fulfilled by NAAG except for the demonstration of its release from nervous tissue on depolarization. For quantification of NAAG in superfusates of brain slices, we have developed an analytical procedure consisting of an ion exchange prepurification, followed by a derivatization procedure and gas chromatography‐mass spectrometry with chemical ionization and selected ion monitoring. Deuterated NAAG was used as an internal standard to provide a high degree of reliability for the analytical method. Detection limits of <1 pmol were achieved. A statistically highly significant increase of NAAG concentration in superfusates from rat neocortex, piriform cortex/amygdala, and hippocampus on depolarization with 50 mM K+ could be demonstrated and was shown to be largely Ca2+ dependent. These results support the hypothesis that NAAG is a neurotransmitter. Especially with respect to the piriform cortex, the present demonstration of NAAG release is consistent with electrophysiological and immunohistochemical evidence for its neurotransmitter function at terminals of the lateral olfactory tract.
Zinc Ions Stabilise the Association of Basic Protein with Brain Myelin Membranes
Authors: Earl Christopher , Chantry Andrew , Mohammad Nazar , Glynn Paul
Published: October 2006

Abstract: Myelin basic protein (MBP) dissociated from brain myelin membranes when they were incubated (37°C; pH 7.4) at physiological ionic strength. Zinc ions inhibited, and calcium promoted, this process. Protease activity in the membrane preparations cleaved the dissociated MBP into both small (<4 kilodaltons) and large (>8 kilodaltons) fragments. The latter were detected, together with intact MBP, by gel electrophoresis of incubation media. Zinc ions appeared to act in two distinct processes. In the presence or absence of added CaC12, zinc ions in the range 0.1–1 mM inhibited MBP‐membrane dissociation. This process was relatively insensitive to heat and Zn2+ could be substituted by either copper (II) or cobalt (II) ions. A second effect was evident only in the presence of added calcium ions, when lower concentrations of Zn2+ (<0.1 mM) inhibited MBP‐membrane dissociation and the accumulation of intact MBP in incubation media. This process was heat sensitive and only copper (II), but not cobalt (II), ions could replace Zn2+. To determine whether endogenous zinc in myelin membranes is bound to MBP, preparations were solubilised in buffers containing Triton X‐100/2 mM CaCl2 and subjected to gel filtration. Endogenous zinc, as indicated by a dithizone‐binding method, eluted with fractions containing both MBP and proteolipid protein (PLP). Thus, one means whereby zinc stabilises association of MBP with brain myelin membranes may be by promoting its binding to PLP.
Selective Protection of Benzomorphan Binding Sites Against Inactivation by N‐Ethylmaleimide. Evidence for k‐Opioid Receptors in Frog Brain
Authors: Zawilska Jolanta , Lajtha Abel , Borsodi Anna
Published: October 2006

Abstract: Selective binding of [3H]bremazocine and [3H]‐ethylketocyclazocine to k‐opioid receptor sites in frog (Rana esculenta) brain membranes is irreversibly inactivated by the sulfhydryl group alkylating agent N‐ethylmale‐imide (NEM). Pretreatment of the membranes with k‐selective compounds [ethylketocyclazocine (EKC), dy‐norphin (1–13), or U‐50,488H] but not with [D‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin (DAGO; μ specific ligand) or [d‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin (DADLE; δ specific ligand) strongly protects the binding of the radioligands against NEM inactivation. These results provide more evidence for the existence of k‐opioid receptors in frog brain. The relatively high concentrations of NEM that are needed to decrease the specific binding of [3H]bremazocine together with the observation of an almost complete protection of its binding sites by NaCl suggest that bremazocine may act as an opioid antagonist in frog brain.
Comparative Histochemical and Biochemical Analysis of Endogenous Receptors for Glycoproteins in Human and Pig Peripheral Nerve
Authors: Gabius Hans‐J. , Kohnke Beatrix , Hellmann Thea , Dimitri Thomas , Bardosi Attila
Published: October 2006

Abstract: Endogenous sugar‐binding proteins were localized in sections of human and pig peripheral nerves by the application of two types of labelled ligands: neoglycopro‐teins (chemically glycosylated carrier proteins that had proven to be histochemically inert) and desialylated, naturally occurring glycoproteins. These proteins allowed evaluation of the presence and distribution of endogenous receptors for carbohydrates, commonly present in cellular glycoconjugates. (Neo)glycoprotein binding was similar, but not identical, for the two types of mammalian peripheral nerves. The pig nerve differed from the human nerve in more pronounced staining when using different types of β‐galactoside‐terminated (neo)glycoproteins and charge‐carrying neoglycoproteins, such as bovine serum albumin, bearing galactose‐6‐phosphate residues, glucuronic acid residues, and sialic acid residues. Comparative biochemical analysis of certain classes of sugar receptors by affinity chromatography and gel electrophoresis revealed the presence of sugar receptors that can contribute to the histochemical staining in a pattern with certain significant differences among rather similar expression for the two species. The assessment of sugar receptor distribution by application of (neo)glycoprotein binding among morphologically defined regions in nerves may hold promise in detecting developmental regulation and changes during nerve degeneration and subsequent regeneration after trauma or pathological states. Correlation of these results to changes in the structure and abundance of glycoconjugates, which are the potential physiological ligands of endogenous sugar receptors commonly detected by plant lectins, may help to infer functional relationships.
Effect of 2‐(4‐Phenylpiperidino)cyclohexanol (AH 5183) on the Veratridine‐Induced Increase in Acetylcholine Synthesis by Rat Hippocampal Tissue
Authors: Carroll P. T. , Ivy M. T.
Published: October 2006

Abstract: The intent of this study was to determine whether the drug 2‐(4‐phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine‐induced increase in acetylcholine (ACh) synthesis by reducing the veratridine‐induced activation of a detergent‐soluble choline‐O‐acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle‐bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by l‐AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of l‐AH 5183 (75 nM), the depolarization‐induced activation of a detergent‐soluble ChAT fraction associated with a vesicle‐bound store of ACh was blocked. Conversely, the veratridine‐induced activation of a water‐soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle‐bound store with newly synthesized ACh following veratridine‐induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergentsoluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183‐sensitive regulatory protein.
Effect of Taurine on Calcium Accumulation in Resting and Depolarised Insect Synaptosomes
Authors: Whitton Peter S. , Nicholson Russell A. , Strang Robin H. C.
Published: October 2006

Abstract: The effect of taurine (2‐aminoethanesulphonic acid) on 45Ca2+ accumulation in resting and depolarised synaptosomes obtained from the locust Schistocerca americana gregaria was studied. Taurine reduced 45Ca2+ accumulation in resting synaptosomes, and this effect was more pronounced when synaptosomes were depolarised with either high [K+] or veratridine. Veratridine‐induced 45Ca2+ accumulation was not affected by either γ‐aminobutyric acid or leucine, but was reduced by both verapamil and tetrodotoxin.
Solubilization and Characterization of a [3H]Hemicholinium‐3 Binding Site in Rat Brain
Authors: Yamada Kiyofumi , Saltarelli Mario D. , Coyle Joseph T.
Published: October 2006

Abstract: A sodium‐dependent high‐affinity [3H]‐hemicholinium‐3 ([3H]HCh‐3) binding site was solubilized from rat striatal synaptic plasma membranes by 0.2% deoxycholate. Deoxycholate solubilization of the [3H]HCh‐3 binding site was dependent upon both detergent concentration and ionic strength of the solubilization medium. Specific [3H]HCh‐3 binding to the solubilized preparation was both sodium‐ and chloride‐dependent and saturable, exhibiting an affinity of 14.2 nM and a capacity (Bmax) of 695 fmol/mg protein. Choline and other analogs inhibited specific [3H]HCh‐3 binding to the solubilized preparation in a concentration‐dependent manner with the similar rank order of potency observed in crude synaptic membranes. Treatments known to disrupt both protein and lipid moieties resulted in diminished specific [3H]HCh‐3 binding. These results suggest that the characteristics of the solubilized [3H]HCh‐3 binding site are similar to those of the membrane‐bound site.
Characterization of the Inhibitory Action of Botulinum Neurotoxin Type A on the Release of Several Transmitters from Rat Cerebrocortical Synaptosomes
Authors: Ashton Anthony C. , Dolly J. Oliver
Published: October 2006

Abstract: Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit ∼90% of Ca2+‐dependent K+‐evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose‐response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at ∼10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+‐independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+‐dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H‐catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4‐aminopyridine could reverse the toxin‐induced blockade of evoked release. This lack of specificity in its action on synaptosomes. and other published findings, lead to the conclusion that toxin‐sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.
Developmental Expression of Neural Cell Adhesion Molecules of Oligodendrocytes In Vivo and in Culture
Authors: Bhat Shama , Silberberg Donald H.
Published: October 2006

Abstract: Previously, we have shown that oligodendrocyte adhesion molecules are related to the 120,000–Mr neural cell adhesion molecule (NCAM‐120). In this report, we present further evidence that the oligodendrocyte adhesion molecule is NCAM‐120. Studies on the expression of NCAM‐120 and other molecular forms of NCAM in vivo in rat brain, in vitro in primary mixed cultures, and in cultures enriched for oligodendrocytes are described. Western blot analysis of rat brain using anti‐NCAM showed that NCAM‐120 first appears at postnatal day 7 and increases in quantity thereafter, coincident with the development of oligodendrocytes in vivo and comparable to the expression of myelin basic protein. Purified oligodendrocytes from 4‐week‐old rat brains expressed only NCAM‐120. Quantitation of various forms of NCAMs in rat brain showed marked age‐related differences in the expression of three molecular forms of NCAM. Immunofluorescence analysis showed that oligodendrocytes, at all ages tested, expressed NCAM, but in older oligodendrocytes, the intensity of staining was less. Western blot analysis of oligodendrocyte‐enriched cultures showed that from day 1 after isolation (12 days of age) through day 7 after isolation (18 days of age) only NCAM‐120 is seen. A possible role for NCAM in myelination and remyelination is discussed.
Guanine Nucleotide Regulation of [125I]β‐Endorphin Binding to Rat Brain Membranes: Monovalent Cation Requirement
Authors: Selley Dana E. , Tyler Corinne B. , Bidlack Jean M.
Published: October 2006

Abstract: The binding of [125I]βh‐endorphin to rat brain membranes was investigated in the presence of GTP and guanylyl‐5′‐imidodiphosphate. In contrast to the binding of the μ‐selective opioid agonist, [3H][d‐Ala2,MePhe4,Glyol5]enkephalin, and the δ‐selective opioid agonist, [3H][d‐peniciIlamine2,d‐penicillamine5]enkephalin, [125I]βh‐endorphin binding was not affected by GTP or guanylyl‐5′‐imidodiphosphate in a concentration‐dependent manner in the absence of cations. However, in the presence of NaCl, the inclusion of either GTP or guanylyl‐5′‐imidodiphosphate resulted in a concentration‐dependent inhibition of [125I]βh‐endorphin binding. This inhibition was significantly greater than the decrease in [125I]βh‐endorphin binding observed in the presence of sodium alone. Although GTP most potently inhibited [125I]βh‐endorphin binding in the presence of sodium, inhibition of [125I]βh‐endorphin binding by GTP was also observed in the presence of the monovalent cations lithium and potassium, but not the divalent cations magnesium, calcium, or manganese. The effect produced by GTP in the presence of NaCl was mimicked by GDP, but not by GMP or other nucleotides. Unlike [125I]βh‐endorphin, the binding of the putative σ receptor agonist, (+)‐[3H]SKF 10,047, was not significantly altered by GTP or guanylyl‐5′‐imidodiphosphate in the absence or presence of sodium.
Evidence for Disulfide Bonds in Membrane‐Bound and Solubilized Opioid Receptors
Authors: Kamikubo Keita , Murase Hiroshi , Murayama Masanori , Matsuda Masafumi , Miura Kiyoshi
Published: October 2006

The effects of pretreatment with dithiothreitol (DTT) on opioid binding activities of membrane‐bound and digitonin‐solubilized opioid receptors from bovine adrenal medulla were studied. Pretreatment of membranes with DTT or mercaptoethanol inhibited [3H]diprenorphine binding by reducing the number of binding sites. The inhibitory action of DTT was time and dose dependent. The binding of [3H]D‐Ala2‐D‐Leu5‐enkephalin was also inhibited by DTT pretreatment. Pretreatment of digitonin‐solubilized binding sites with DTT also reduced the number of [3H]diprenorphine binding sites. The action of DTT was diminished by preincubating the DTT solution with H2O2, [3H]Diprenorphine protected the opioid binding sites from the inhibitory action of DTT. The present results provide evidence that disulfide bonds are implicated in opioid binding activity of the opioid receptor system.
Effect of Forskolin and Prostaglandin E1 on Stimulus Secretion Coupling in Cultured Bovine Adrenal Chromaffin Cells
Authors: Marriott Deborah , Adams Maria , Boarder Michael R.
Published: October 2006

Treatment of adrenal chromaffin cells with forskolin (0.1–10 μM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20–17–24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine‐induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine‐stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium‐ and nicotine‐stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura‐2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 μM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.
Hypoxanthine Transport and Metabolism in the Central Nervous System
Authors: Spector Reynold
Published: October 2006

The mechanisms by which hypoxanthine, the principal purine in plasma and CSF, enters and leaves rabbit brain, choroid plexus, and CSF were investigated in the isolated choroid plexus in vitro and by injecting [14C]hypo‐xanthine intraventricularly and [3H]hypoxanthine intravenously. The isolated choroid plexus accumulated and extensively metabolized [I4C]hypoxanthine; however, l4C was readily released from choroid plexus principally as [I4C]‐hypoxanthine. After infusion of [3H]hypoxanthine intravenously, [3H]hypoxanthine entered CSF and brain slowly and was converted in brain to nucleotides. Fewer than 5% of the acid‐soluble purine nucleotides in brain entered rabbit brain from plasma hypoxanthine (and inosine) per 24 h. After intraventricular injection of [l4C]hypoxanthine, the [14C]hypoxanthine was cleared from the CSF into the blood or accumulated by brain and largely converted into 14C‐nucleotides. Little [14C]xanthine and no [14C]uric acid or allantoin were formed. These studies show that brain, unlike most other tissues, rapidly recycles hypoxanthine and converts it into purine nucleotides, and not unsalvageable purines.Key Words: Blood‐brain barrier—Cerebrospinal fluid—Choroid plexus. Spector R. Hypoxanthine transport and metabolism in the central nervous system.
A Putative M3 Muscarinic Cholinergic Receptor of High Molecular Weight Couples to Phosphoinositide Hydrolysis in Human SK‐N‐SH Neuroblastoma Cells
Authors: Fisher Stephen K. , Heacock Anne M.
Published: October 2006

The M1‐selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N‐[3H]methylscopolamine ([3H]NMS) and [3H]qui‐nuclidinylbenzilate from intact human SK‐N‐SH neuroblastoma cells with a low affinity (Ki= 869–1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF‐DX 116 {11–2[[2‐[(diethylamino)methyl]‐1‐piperidinyl]‐acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4]benzodiazepin‐6‐one} bound to the mAChRs with a very low affinity (Ki= 6.0 μM), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki= 7.2 nM). 4‐DAMP was also 1,000‐fold more effective than AF‐DX 1 16 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilylcholine mustard) suggest that the size of the SK‐N‐SH mAChR (Mr= 81.000–98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr=66,000), an M1‐enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.
A Comparison of Methodologies for the Study of Functional Transmitter Neurochemistry in Human Brain
Authors: Dodd Peter R. , Hambley John W. , Cowburn Richard F. , Hardy John A.
Published: October 2006

Abstract: A number of different approaches to the study of functional neurochemistry in human brain are discussed. The advantages and disadvantages of three main techniques are contrasted: (i) using animal tissue preparations as models of the human brain; (ii) using human peripheral tissue preparations as models of dynamic CNS processes; and (iii) studying human tissue, obtained postmortem, directly. Animal models are often readily obtained and reliable, and the high degree of inbreeding of common laboratory animals ensures that they usually yield consistent results. However, there are a number of human disorders for which animal models are either poor or unavailable, and species differences make extrapolation from the animal to the human case difficult. Human peripheral tissue models rely on a degree of homology between peripheral and CNS processes; in most cases, the evidence for such homologies derives from animal, rather than human, studies. Moreover, several examples are known where a peripheral process mimics the equivalent glial cell activity more closely than the neuronal, which can be a serious drawback for studies of neurotransmission. The use of postmortem human brain tissue presents a number of obvious difficulties, resulting from variations in the patient's age, agonal state, sex, preterminal medication, postmortem delay, etc. Human beings are genetically and nutritionally heterogeneous, so that data variability is usually greater here than when using tissue from laboratory animals. However, it is possible to control for a number of these factors, for example, by matching samples for basal metabolic rate and tissue integrity, and recently developed tissue freezing and storage techniques permit the use of within‐subject experimental designs to help reduce experimental variation. A range of neurotransmitter functions are well retained in such tissue samples, so that regional variations, differential transmitter activities, drug effects, etc., can be studied in normal tissue samples, as well as in samples taken from cases of neurological and psychiatric disease. This allows, for example, changes in neuroanatomical indices to be correlated with localised alterations in a specific neurotransmitter function. A systematic approach to the analysis and matching of tissue samples is advocated. The three approaches should be considered to be complementary, especially for the study of human brain diseases.
Cholinergic Surface Antigen Chol‐1 Is Present in a Subclass of VIP‐Containing Rat Cortical Synaptosomes
Authors: Agoston Denes V. , Borroni Edilio , Richardson Peter J.
Published: October 2006

Abstract: The colocalization of vasoactive intestinal polypeptide (VIP) with the cholinergic specific surface antigen Chol‐1 was investigated in synaptosomes derived from the rat cerebral cortex. Immunoaffinity purification of cortical synaptosomes using antisera to Chol‐1 resulted in the copurification of VIP and cholinergic nerve terminals. VIP was purified with a yield of 75% of that of choline acetyltransferase (ChAT). These results suggest that approximately 53% of the cortical cholinergic terminals contain VIP, whereas 75% of the cortical VIP content is present in these cholinergic terminals. Both hypotonic lysis and depolarization of the nerve terminals resulted in the differential release of VIP and acetylcholine (ACh), indicating the different compartmentalization in the same nerve terminal. Complement‐mediated lysis of cholinergic nerve terminals, using antisera to Chol‐1, resulted in the release of 64% of the ChAT, 71% of ACh, and 27% of the VIP. The application of our method enables quantifying and mapping, with a fast, efficient, and specific technique, the coexisting peptides in cholinergic neurons of distinct brain areas.
Effects of Ammonia and β‐Methylene‐dl‐Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture
Authors: Fitzpatrick Susan M. , Cooper Arthur J. L. , Hertz Leif
Published: October 2006

Abstract: Both ammonia and β‐methylene‐dl‐aspartate (β‐MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate‐aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and β‐MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate‐aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mMβ‐MA resulted in a decreased production of 14CO2 from [U‐14Clglucose, but did not affect 14CO2 production from [2–14C] pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2–14C] pyruvate, but 14CO2 production from [U‐14C] glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U‐14C] glucose or [2–14]pyruvate by neurons. However, incubation of neurons with β‐MA or β‐MA plus ammonium chloride resulted in a 45% decrease of 14CO2 production from both [U‐14C] glucose and [2–14C] pyruvate. A 2‐h incubation of astrocytes with β‐MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in >50% decrease in ATP, but had little effect on phosphocreatine. β‐MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards β‐MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by ∼45% in astrocytes and by ∼55% in neurons.
Measurement of Free Intracellular Calcium in the Brain by 19F‐Nuclear Magnetic Resonance Spectroscopy
Authors: Bachelard H. S. , Badar‐Goffer R. S. , Brooks K. J. , Dolin S. J. , Morris P. G.
Published: October 2006

Abstract: We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F‐nuclear magnetic resonance calcium chelator, 5,5′‐F2‐1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.
Polyamines, Ornithine Decarboxylase, and Diamine Oxidase in the Substantia Nigra and Striatum of the Male Rat After Hemitransection
Authors: Desiderio Maria Alfonsina , Zini Isabella , Davalli Pierpaola , Zoli Michele , Corti Arnaldo , Fuxe Kjell , Agnati Luigi Francesco
Published: October 2006

Partial hemitransection at the mesodiencephalic junction in the rat increased striatal and nigral putrescine concentrations on the lesioned side for at least 168 h, with maximal increases between 24 and 48 h. Spermidine and spermine levels declined at 24 h in the striatum, rising above control values at 48 h and further at 168 h. In the substantia nigra, they remained unchanged for the first 48 h and then increased by 168 h. Cadaverine in the striatum also increased at 48 h. On the intact side putrescine increased but to a much lesser extent (at 48 h in the striatum and at 24 and 48 h in the substantia nigra). Ornithine decarboxylase and diamine oxidase activities showed maximal increases at 24 h in the striatum of the lesioned side, whereas in the substantia nigra ornithine decarboxylase attained a very high value as early as 4 h after the operation and diamine oxidase activity peaked at 48 h. The enzyme activities returned toward the basal values at 168 h. On the intact side, ornithine decarboxylase showed a small increase starting at 4 h and diamine oxidase was enhanced at 48 h. These results indicate that the stimulation of biosyn‐thetic and degradative enzymes of polyamine metabolism accompanied by marked and prolonged increases in putrescine may be essential events in the early phases of neuronal response to mechanical injury in the CNS.
Solubilization of Rat Brain Phencyclidine Receptors in an Active Binding Form That Is Sensitive to N‐Methyl‐d‐Aspartate Receptor Ligands
Authors: Ambar Ifat , Kloog Yoel , Sokolovsky Mordechai
Published: October 2006

Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20–30% of the high‐affinity PCP binding sites were solubilized. The high affinity toward PCP‐like drugs, the stereo‐selectivity of the sites, and the sensitivity to N‐methyl‐d‐aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N‐[3H][l‐(2‐thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD= 35 nM), and PCP‐like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane‐bound receptors; the most potent inhibitors were TCP (Ki= 31 nM) and the anticonvulsant MK‐801 (Ki= 50 nM). The NMDA receptor antagonist 2‐amino‐5‐phosphonovaleric acid inhibited‐binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose‐dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.
Monoamine Oxidase‐Inhibiting Properties of SR 95191, a New Pyridazine Derivative, in the Rat: Evidence for Selective and Reversible Inhibition of Monoamine Oxidase Type A In Vivo but Not In Vitro
Authors: Kan Jean‐Paul , Steinberg Régis , Leclercq Jérome , Worms Paul , Biziere Kathleen
Published: October 2006

Abstract: In rodents, SR 95191 [3‐(2‐morpholinoethylamino)‐4‐cyano‐6‐phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short‐acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO‐A and MAO‐B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO‐A (ED50= 3–5 mg/kg, p.o.), whereas MAO‐B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1–100 mg/kg, p.o.) antagonized, in a dose‐dependent fashion, the irreversible inhibition of brain and liver MAO‐A induced by phenelzine. Finally, dopamine and 5‐hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO‐A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1–10 mg/kg) towards MAO‐A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO‐A, but not MAO‐B, by SR 95191 was time dependent, with a 19‐fold decrease in the IC50 values being observed over a 30‐min incubation period (140 to 7.5 μM). At this time, the SR 95191 ‐induced inhibition of MAO‐A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO‐A was fully competitive (Ki= 68 μM). It is concluded that SR 95191 is a selective and reversible type A MAOI in vivo but not in vitro.
Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester
Authors: Neary J. T. , Norenberg L. O. B. , Norenberg M. D.
Published: October 2006

Abstract: The distribution of calcium‐activated, phospholipid‐dependent protein kinase (protein kinase C) in supernatant and paniculate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine‐rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30–60 min) of the cultures with 100 nM 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), but not with 4α‐phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphor ylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two‐thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well‐suited to respond to signals that activate phosphoinositide‐linked receptors in brain.
Nicotinic Agonists Regulate α‐Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells
Authors: Siegel Hal N. , Lukas Ronald J.
Published: October 2006

Abstract: The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane‐bound high‐affinity binding sites for α‐bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d‐tubocurarine, induced up to a fivefold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co‐incubation with the nicotinic antagonists d‐tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10–13 h lag phase upon removal of agonist, recovery of the up‐regulated sites to control values occurred within an additional 10–20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine‐induced up‐regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine‐induced receptor blockade does not appear to be involved.
Release of Endogenous Taurine and γ‐Aminobutyric Acid from Brain Slices from the Adult and Developing Mouse
Authors: Oja Simo S. , Kontro Pirjo
Published: October 2006

Abstract: The spontaneous and potassium‐stimulated release of endogenous taurine and γ‐aminobutyric acid (GABA) from cerebral cortex and cerebellum slices from adult and developing mice was studied in a superfusion system. The spontaneous release of GABA was of the same magnitude in slices from adult and developing mice, but the spontaneous release of taurine was considerably greater in the adults. The potassium‐stimulated release of GABA from cerebral cortex slices was about five times greater in adult than in 3‐day‐old mice, but the potassium‐stimulated release of taurine was more than six times greater in 3‐day‐old than in adult mice. In cerebellar slices from 7‐day‐old mice, potassium stimulation also evoked a massive release of taurine, whereas the evoked release from slices from adult mice was rather negligible. Also in cerebellar slices the potassium‐stimulated release of GABA exhibited the opposite quantitative pattern. The stimulated release of both GABA and taurine was partially calcium dependent. The results suggest that taurine may be an important regulator of excitability in the developing brain.
Glutamate Stimulation of [3H]Dopamine Release from Dissociated Cell Cultures of Rat Ventral Mesencephalon
Authors: Mount H. , Welner S. , Quirion R. , Boksa P.
Published: October 2006

Abstract In dissociated cell cultures of fetal rat ventral mesencephalon preloaded with [3H]dopamine, glutamate (10‐5‐10‐3M) stimulated the release of [3H]dopamine. Glutamate stimulation of [3H]dopamine release was Ca2+ dependent and was blocked by the glutamate antagonist, cis‐2,3‐piperidine dicarboxylic acid. Glutamate stimulation of [3H]dopamine release was not due to glutamate neurotoxicity because (1) glutamate did not cause release of a cytosolic marker, lactate dehydrogenase, and (2) preincubation of cultures with glutamate did not impair subsequent ability of the cells to take up or release [3H]dopamine. Thus, these dissociated cell cultures appear to provide a good model system to characterize glutamate stimulation of dopamine release. Release of [3H]dopamine from these cultures was stimulated by verat‐ridine, an activator of voltage‐sensitive Na+ channels, and this stimulation was blocked by tetrodotoxin. However, glutamate stimulated [3H]dopamine release was not blocked by tetrodotoxin or Zn2+. Substitution of NaCl in the extracellular medium by sucrose, LiCl, or Na2SO4 had no effect on glutamate stimulation of [3H]dopamine release; however, release was inhibited when NaCl was replaced by choline chloride or N‐methyl‐d‐glucamine HCl. Glutamate‐stimulated [3H]‐dopamine release was well maintained (60‐82% of control) in the presence of Co2+, which blocks Ca2+ action potentials, and was unaffected by the local anesthetic, lidocaine. These results are discussed in terms of the receptor and ionic mechanisms involved in the stimulation of dopamine release by excitatory amino acids.
L‐[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle
Authors: Syvertsen Christian , Fonnum Frode
Published: October 2006

Abstract: The binding of L‐[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L‐[3H]glutamate. The preparation showed high specific binding of L‐glutamate with a KD of 0.12 μM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4°C. Nonspecific binding was 5–10% of total binding. The gluta‐mate binding was highly stereospecific [K0.5 (D‐glutamate), 270 μM] and showed a high degree of discrimination between l‐glutamate and l‐aspartate [K0.5 (L‐aspartate), 54 μM]. In mammalian CNS preparations potent agonists of l‐glutamate such as kainate and N‐methyl‐d‐aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of l‐glutamate binding [K0.5 (quisqualate), 162 μM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 μM], and various esters of l‐glutamate were of intermediate potency as displacers of l‐[3H]glutamate binding (K0.5 values from 6 to 60 μM) The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra‐junctional or a junctional receptor.