bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats
Authors: Nagata Yutaka , Ando Masato , Takahama Kazuya , Iwata Mitsuyoshi , Hori Shinichiro , Kato Kanefusa
Published: October 2006

Abstract: : The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two‐site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3–9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3–6 h after operation, and even unilateral re moval of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi‐than iN the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did re moval of the glands. These results suggest that the endoge nous NGF that accumulates in the SCG is mostly synthe sized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.
In Situ Molecular Size of Agonist Dopamine D‐2 Binding Sites in Rat Striatum
Authors: Gredal Ole , Nielsen Mogens
Published: October 2006

Abstract: Specific binding of [3H]N‐propylnorapomorphine ([3H]NPA) to 3,4‐dihydroxyphenylethylamine (dopamine) D‐2 receptors was investigated in rat striatum in vitro. For various dopamine receptor substances, the rank order of potency to inhibit [3H]NPA binding was spiroperidol ≧NPA ≥ LY 171555 ≥ SCH 23390 ≥ SKF 38393. A single high‐affinity binding site was found in membranes prepared in either Tris‐citrate buffer or imidazole buffer; the affinity constants were 0.11 and 0.76 nM, respectively. The number of receptors (33 pmol/g wet weight) was independent of whether the membranes were prepared in Tris‐citrate buffer or imidazole buffer and was similar to the number of receptors estimated by [3H]spiroperidol binding to dopamine receptors. Irradiation inactivation of frozen whole rat striata showed a monoexponential loss of [3H]NPA binding sites without a change in the binding affinity. The target size of the [3H]NPA binding site was 81,000 daltons, which shows that the functional molecular entity to bind the dopamine D‐2 agonist was smaller than the molecular entity to bind the dopamine D‐2 antagonist [3H]spiroperidol (target size, 137,000 daltons).
Dementia of the Alzheimer's Type: Changes in Hippocampal L‐[3H]Glutamate Binding
Authors: Greenamyre J. Timothy , Penney John B. , D'Amato Constance J. , Young Anne B.
Published: October 2006

Abstract: Glutamate or a related excitatory amino acid is thought to be the major excitatory neurotransmitter of hippocampal afferents, intrinsic neurons, and efferents. We have used an autoradiographic technique to investigate the status of excitatory amino acid receptors in the hippocampal formation of patients dying with dementia of the Alzheimer type (DAT). We examined l‐[3H]glutamate binding to sections from the hippocampal formation of six patients dying of DAT and six patients without DAT and found marked reductions in total [3H]glutamate binding in all regions of hippocampus and adjacent parahippocampal cortex in DAT brains as compared to controls. When subtypes of excitatory amino acid receptors were assayed, it was found that binding to the N‐methyl‐d‐aspartate (NMDA)‐sensitive receptor was reduced by 75–87%, with the greatest loss found in stratum moleculare and stratum pyramidale of CA1. Binding to quisqualate (QA)‐sensitive receptors was reduced by 45–69%. There were smaller reductions (21–46%) in GABAA receptors in DAT cases. Muscarinic cholinergic receptors assayed in adjacent sections of hippocampal formation were unchanged in DAT. Benzodiazepine receptors were reduced significantly only in parahippocampal cortex by 44%. These results suggest that glutamatergic neurotransmission within the hippocampal formation is likely to be severely impaired in Alzheimer's disease. Such impairment may account for some of the cognitive decline and memory deficits that characterize DAT.
Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets
Authors: Meyerson Laurence R. , Ieni John R. , Wennogle Lawrence P.
Published: October 2006

Abstract: The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5‐hydroxytryptamine (5‐HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N‐ethylmaleimide (NEM) differentially affected [3H]5‐HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5‐HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5‐HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5‐HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216,303, pyril‐amine, and bicifadine were preferential [3H]5‐HT uptake blockers. 5‐HT was a weak displacer of [3H]IMI binding (IC25= 3.0 μM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5‐HT transporter complex in human platelets.
Distribution of Putrescine in Rat Brain Measured by Gas Chromatography‐Mass Spectrometry
Authors: Noto Tadashi , Hasegawa Takeshi , Hashimoto Hiromichi , Nakajima Teruo
Published: October 2006

Abstract: We measured putrescine levels in minute sites of single rat brains using a sensitive, specific assay involving gas chromatography‐mass spectrometry. The putrescine level was measured in 20 sites of single rat brains: three sites in the cerebral cortex, six sites in the hypothalamus, three sites in the basal ganglia, three sites in the thalamus, three sites in the limbic system, and two sites in the cerebellum. The level of putrescine was very high in the hypothalamus, high in the basal ganglia and limbic system, and low in the thalamus, cerebellum, and two of the three sites in the cerebral cortex. The highest levels were in the anterior hypothalamic area and the lateral hypothalamic area, and the lowest levels were in the vermis and the lobe of the cerebellum.
Tyrosine Hydroxylase, Tryptophan Hydroxylase, Biopterin, and Neopterin in the Brains of Normal Controls and Patients with Senile Dementia of Alzheimer Type
Authors: Sawada Makoto , Hirata Yoko , Arai Heii , Iizuka Reiji , Nagatsu Toshiharu
Published: October 2006

Abstract: The activities of tyrosine hydroxylase and tryptophan hydroxylase, and the concentrations of the biopterin cofactor and the precursor neopterin were measured in 14 regions of postmortem brains from four histologically verified patients of senile dementia of the Alzheimer type (SDAT) and eight histologically normal controls. Neopterin concentrations were measured in the human brain for the first time. The activities of tyrosine hydroxylase and tryptophan hydroxylase in the brains of patients with SDAT were significantly reduced in the substantia nigra and in the lateral segment of the globus pallidus, locus ceruleus, and substantia nigra, respectively. The concentrations of total biopterin in the brains of patients with SDAT were signifycantly reduced in the putamen and substantia nigra, but the total neopterin concentrations did not change significantly. These results suggest that the reduction in biogenic amines in SDAT might be related to reductions in biosynthetic enzymes associated with biogenic amines, due to destruction of monoaminergic neurons.
Uncoupling of Cerebral Glucose Supply and Utilization After Hexane‐2,5‐Dione Intoxication in the Rat
Authors: Planas Anna M. , Cunningham Vincent J.
Published: October 2006

Abstract: Chronic administration of hexane‐2,5‐dione (2,5‐HD) to rats causes an accumulation of neurofilaments within axons that may lead to their degeneration. This occurs in both the CNS and PNS. It has been suggested that one of the effects of 2,5‐HD is an impairment of glucose utilization arising from an inhibition of specific glycolytic enzymes. This hypothesis is based principally on evidence obtained in vitro. In the present study, glucose utilization, glucose transport across the blood‐brain barrier, and blood flow have been measured in vivo in brain regions of control rats and in three groups of rats treated with 2,5‐HD as (a) a single intragastric dose (500 mg/kg of body weight), (b) high chronic doses of 500 mg/kg of body weight for 15 days, or (c) low chronic doses of 250 mg/kg of body weight for 21 days. Group b showed overt signs of neuropathy, whereas groups a and c did not. The results indicate two independent effects of 2,5‐HD in the CNS: a dose‐dependent inhibition of glucose utilization and an effect on glucose supply and transport across the blood‐brain barrier, which is apparent only after chronic treatment.
Neurochemical and Immunocytochemical Studies on the Distribution of N‐Acetyl‐Aspartylglutamate and N‐Acetyl‐Aspartate in Rat Spinal Cord and Some Peripheral Nervous Tissues
Authors: Ory‐Lavollée L. , Blakely R. D. , Coyle J. T.
Published: October 2006

Abstract: HPLC analysis of rat spinal cord revealed a uniform distribution of N‐acetyl‐aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N‐acetyl‐aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity‐purified antiserum raised against NAAG‐bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.
Raised Thyrotrophin‐Releasing Hormone, Pyroglutamylamino Peptidase, and Proline Endopeptidase Are Present in the Spinal Cord of Wobbler Mice but Not in Human Motor Neurone Disease
Authors: Court J. A. , McDermott J. R. , Gibson A. M. , Marshall E. , Bloxham C. A. , Perry R. H. , Edwardson J. A.
Published: October 2006

Abstract: The Wobbler mouse (wr) is a mutant that exhibits loss of anterior horn ceils in the spinal cord and brainstem and subsequent muscle wasting, particularly of the fore‐limbs and neck. The wr mice, 2–3 months of age, were found to have increased levels of immunoreactive‐thyrotrophin‐releasing hormone (ir‐TRH) in the spinal cord and pons and medulla, but not in other CNS areas. This increase was observed in dorsal and ventral cord and at cervical, thoracic, and lumbar levels and was confirmed by HPLC to be authentic TRH. The levels of immunoreactive‐somatostatin,‐neurotensin, and‐substance P were not raised in the CNS of wr mice. The activities of two peptidases capable of degrading TRH, pyroglutamylaminopeptidase (PGAP, EC 3.4.11.8) and proline endopeptidase (PEP, EC 3.4.21.26), and the level of 5‐hydroxyindoleacctic acid were also raised in the spinal cord of 2–3‐month‐old wr mice although the activities of alanine aminopeptidase and lactate dehydrogenase and the level of 5‐hydroxytrypt‐amine were not. Increased spinal cord levels of ir‐TRH and PGAP and PEP activities were not observed in the 1‐month‐old wr mice. In addition, a pilot study using spinal cord obtained at autopsy from three patients with motor neurone disease and 12 control subjects indicated no increase in spinal cord ir‐TRH, PGAP, or PEP in human motor neurone disease.
Sub cellular Fractionation of the Longitudinal Smooth Muscle/Myenteric Plexus of Dog Ileum: Dissociation of the Distribution of Two Plasma Membrane Marker Enzymes
Authors: Kostka P. , Ahmad S. , Berezin I. , Kwan C. Y. , Allescher H. D. , Daniel E. E.
Published: October 2006

Abstract: The distribution of plasma membrane markers, the sodium pump [evaluated as ouabain‐sensitive, potassium‐stimulated p‐nitrophenyl phosphatase (K+‐pNPPase)], [3H]saxitoxin binding, and 5′‐AMPase, was studied in the subcellular fractions prepared from the ho‐mogenates of the longitudinal smooth muscle/myenteric plexus of dog ileum. The K+‐pNPPase activity and [3H]‐saxitoxin binding were found to be predominantly associated with the synaptosomal fraction as indicated by the high level of these activities in the crude synaptosomal fraction and by the copurification of K+‐pNPPase and [3H]saxitoxin binding, but not 5′‐AMPase, with several synaptosomal markers during the fractionation of the crude synaptosomal fraction on density gradients. In contrast to the K+‐pNPPase activity and [3H]saxitoxin binding, the 5′‐AMPase activity was found to be concentrated in the microsomal pellet. Further fractionation of microsomes on a density gradient resulted in copurification of 5′‐AMPase, but not K+‐pNPPase or [3H]saxitoxin binding, with other smooth muscle plasma membrane‐bound enzymes, such as high‐affinity Ca2+‐ATPase, Mg2+‐ATPase, and Ca2+‐ATP‐ase. It was concluded that in the longitudinal smooth muscle/myenteric plexus, the sodium pump activity is present in higher density in the neuronal plasma membranes whereas 5′‐AMPase activity is concentrated in the smooth muscle plasma membranes.
Perinatal Hypoxic‐Ischemic Brain Injury Enhances Quisqualic Acid‐Stimulated Phosphoinositide Turnover
Authors: Chen Chu‐Kuang , Silverstein Faye S. , Fisher Stephen K. , Statman Daniel , Johnston Michael V.
Published: October 2006

Abstract: In an experimental model of perinatal hypoxic‐ischemic brain injury, we examined quisqualic acid (Quis)‐stimulated phosphoinositide (PPI) turnover in hippocampus and striatum. To produce a unilateral forebrain lesion in 7‐day‐old rat pups, the right carotid artery was ligated and animals were then exposed to moderate hypoxia (8% oxygen) for 2.5 h. Pups were killed 24 h later and Quis‐stimulated PPI turnover was assayed in tissue slices obtained from hippocampus and striatum, target regions for hypoxic‐ischemic injury. The glutamate agonist Quis (10‐4M) preferentially stimulated PPI hydrolysis in injured brain. In hippocampal slices of tissue derived from the right cerebral hemisphere, the addition of Quis stimulated accumulation of inositol phosphates by more than ninefold (1,053 ± 237% of basal, mean ± SEM, n = 9). In contrast, the addition of Quis stimulated accumulation of inositol phosphates by about fivefold in the contralateral hemisphere (588 ± 134%) and by about sixfold in controls (631 ± 177%, p < 0.005, comparison of ischemic tissue with control). In striatal tissue, the corresponding values were 801 ± 157%, 474 ± 89%, and 506 ± 115% (p < 0.05). In contrast, stimulation of PPI turnover elicited by the cho‐linergic agonist carbamoylcholine, (10‐4 or 10‐2M) was unaffected by hypoxia‐ischemia. The results suggest that prior exposure to hypoxia‐ischemia enhances coupling of excitatory amino acid receptors to phospholipase C activity. This activation may contribute to the pathogenesis of irreversible brain injury and/or to mechanisms of recovery.
Excitatory Amino Acid‐Evoked Release of γ‐[3H]Aminobutyric Acid from Striatal Neurons in Primary Culture
Authors: Weiss Samuel
Published: October 2006

Abstract: The actions of excitatory amino acids on the release of previously incorporated γ‐[3H]aminobutyric acid ([3H]GABA) were examined in purified (>93%) striatal neurons derived from the fetal mouse brain and differentiated in primary culture. Glutamate, KC1, and veratrine evoked a dose‐dependent, saturable, and reversible release of [3H]GABA from striatal neurons. Glutamate actions were not reduced in the absence of calcium, and were insensitive to tetrodotoxin. The dose‐response relationships of excitatory amino acids demonstrated the following rank order of potency: glutamate > aspartate =N‐methyl‐d‐aspartate > kainate ≧ quisqualate. Kainate, however, was the most effective agonist, evoking an eightfold increase over baseline levels of [3H]GABA release. Aspartate‐ and N‐methyl‐d‐aspartate‐evoked release was abolished in the presence of either 2‐aminophosphonovaleric acid or γ‐d‐glutamylglycine. Release due to glutamate and kainate was partially or ineffectively attenuated by these agents. Glutamate‐, aspartate‐, and N‐methyl‐d‐aspartate‐evoked GABA releases were augmented when calcium was omitted from the bathing medium and reduced when sodium was replaced with choline or lithium. Kainate‐evoked release was unaffected when calcium was omitted, virtually unchanged when choline replaced sodium, and markedly potentiated when lithium was substituted for sodium. These findings suggest that at least two distinct receptor systems for excitatory amino acids mediate the evoked release of [3H]GABA from striatal neurons in primary culture. These two systems, aspartate/N‐methyl‐d‐aspartate‐ and kainate‐prefer‐ring, are distinguishable on the basis of their pharmacological and ionic properties.
Differential Control by N‐Methyl‐D‐Aspartate and Kainate of Striatal Dopamine Release In Vivo: A Trans‐Striatal Dialysis Study
Authors: Carter C. J. , L'Heureux R. , Scatton B.
Published: October 2006

Abstract: Using the technique of transstriatal dialysis in halothane‐anesthetized rats, we have studied the effects of intrastriatally infused N‐methyl‐D‐aspartate (NMDA), kainate, and quisqualate on the liberation of endogenous striatal dopamine. The striatal infusion of NMDA (10‐3‐10‐2M) or kainate (10‐4‐10‐2M) but not of quisqualate (up to 10‐2M) for one 20‐min fraction provoked a dramatic increase in striatal dopamine efflux up to a maximum of 1,200 and 3,400% of basal levels for NMDA and kainate, respectively. NMDA (10‐3M) evoked liberation of striatal dopamine was totally blocked by coinfusion of 2‐amino‐5‐phosphonovalerate (2‐APV; 5 × 10‐4M) and by the systemic injection of phencyclidine (3 mg/kg i.p.). The effects of NMDA (10‐3M) were also totally antagonized in a dose‐dependent manner by the striatal coinfusion of atropine (10‐7‐10‐4M), and abolished in rats that had received bilateral striatal ibotenate lesions (10μg/1μl) 1 week prior to implantation of the dialysis fiber. The striatal infusion of tetrodotoxin (10‐6M) reduced basal dopamine efflux by 60–70% and abolished the NMDA (10‐3M)‐evoked liberation of striatal dopamine. The effects of kainate (10‐3M) on striatal dopamine efflux were only partially reduced by doses of 2‐APV or atropine that totally blocked the NMDA response, and were also partially resistant to tetrodotoxin. The intrastriatal infusion of carbachol (10‐3M) for one 20‐min fraction was without effect on striatal dopamine efflux; 10‐2M carbachol produced a 50% increase in dopamine release in the fraction following carbachol application. The present results indicate that the effects of NMDA on striatal dopamine release are likely to be mediated via an active cholinergic striatal neuron which seems to play a permissive rather than an active role in this effect. The effects of kainate may involve a direct action on dopa‐minergic terminals as well as activation of polysynaptic pathways or liberation of an endogenous NMDA receptor agonist.
Differences in the Biophysical Properties of the Benzodiazepine/γ‐Aminobutyric Acid Receptor Chloride Channel Complex in the Long‐Sleep and Short‐Sleep Mouse Lines
Authors: McIntyre Todd D. , Trullas Ramon , Skolnick Phil
Published: October 2006

Abstract: Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long‐sleep (LS) and short‐sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55°C) at a significantly faster rate than those from SS mice. Although γ‐aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0° to 37°C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex (“supramolecular complex”), together with a higher KD for t‐[35S]butylbicyclophosphoro‐thionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.
Differential Effects of Tachykinins Injected Intranigrally on Striatal Dopamine Metabolism
Authors: Tan Dong‐Ping , Tsou Kang
Published: October 2006

Abstract: The effects of intranigral injection of kassinin, ele‐doisin, and substance P on striatal dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) contents as well as circling behavior were studied in rats. Kassinin and eledoisin produced a marked dose‐dependent increase of DOPAC concentrations in the ipsilateral striatum, as well as in the number of contralateral circlings. Substance P produced a similar but weaker effect. At the larger dose (5 nmol), the three tachykinins also induced an increase of DA concentrations in the ipsilateral striatum. The rank order of activity was kassinin > eledoisin > substance P. These results suggest that tachykinins stimulated the nigro‐striatal dopaminergic system by accelerating the dopamine metabolism in striatum.
GABAB Receptor‐Mediated Inhibition of Histamine H1‐Receptor‐Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex
Authors: Crawford Melissa L. A. , Young J. Michael
Published: October 2006

Abstract: Histamine‐stimulated accumulation of [3H]inositol monophosphate ([3H]IP,) in lithium‐treated slices of rat cerebral cortex was inhibited by γ‐aminobutyric acid (GABA) (IC50 0.30 ± 0.03 mM). The maximum level of inhibition was 69 ± 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAb agonist (‐)‐baclofen, IC50 0.69 ± 0.04 μM, which was 430‐fold more potent as an inhibitor than the (+)‐isomer. (‐)‐Baclofen also inhibited histamine‐induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (‐)‐and (+)‐baclofen had Hill coefficients greater than unity. (—)‐ Baclofen, at concentrations that caused inhibition of histamine‐induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine‐stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol‐stimulated [3H]IP1 formation.
Purification and Characterization of P400 Protein, a Glycoprotein Characteristic of Purkinje Cell, from Mouse Cerebellum
Authors: Maeda Nobuaki , Niinobe Michio , Nakahira Kensuke , Mikoshiba Katsuhiko
Published: October 2006

Abstract: P400 protein is a concanavalin A (Con A)‐binding, 250–kilodalton glycoprotein characteristic of cerebellum. Extraction conditions for P400 protein were investigated, and complete solubilization of P400 protein from a submicrosomal fraction (P31 fraction) of mouse cerebellum was attained by the combination of 4% Zwittergent 3–14 and 4 M guanidinium chloride. The solubilized P400 protein was purified using Sepharose CL‐4B and Con A‐Sepharose chromatography. A monoclonal antibody (18A10) was prepared against P400 protein. Endo‐β‐N‐acetylglucosaminidase F digestion of P400 protein revealed that P400 protein has a small number of asparagine‐linked oligosaccharide chains and that the epitope that is recognized by 18A10 monoclonal antibody is not on the asparagine‐linked oligosaccharide portion. Tissue distribution of P400 protein was investigated by immunoblot analysis using 18A10 monoclonal antibody. P400 protein was abundant in the cerebellum, but a very small amount of P400 protein or related antigen was also detected in other parts of the nervous system and in nonneural tissues. Immunohis‐tochemical studies indicated that P400 protein was distributed abundantly in the soma, the dendritic arborization, and the axon of the Purkinje cell. No immunoreaction was observed in the other types of cells.
Poly(N‐Acetyllactosaminyl) Oligosaccharides of Chromaffin Granule Membrane Glycoproteins
Authors: Margolis Richard U. , Fischer‐Colbrie Reiner , Margolis Renée K.
Published: October 2006

Abstract: Poly(N‐acetyllactosaminyl) oligosaccharides have been identified, on the basis of their susceptibility to endo‐β‐galactosidase, in a large‐molecular‐size glycopeptide fraction derived from chromaffin granule membrane glycoproteins. The glycoproteins containing poly(N‐acetyl‐lactosaminyl) oligosaccharides were selectively labeled by treatment of chromaffin granule membranes with endo‐β‐galactosidase to expose N‐acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP‐[14C]galactose. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and fluorography demonstrated specific labeling in the 41–47 kilodalton (kD) region and in a distinct band at 90 kDa. Two‐dimensional SDS‐PAGE revealed that the poly(N‐acetyllactosaminyl) oligosaccharides are predominantly present in glycoprotein IV, together with lesser labeling of glycoproteins II and III, whereas they are absent from dopamine β‐hydroxylase and carboxypeptidase H, which are the major glycoproteins of chromaffin granule membranes.
Serotonin 5‐HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein
Authors: Herrick‐Davis Katharine , Titeler Milt , Leonhardt Sigrun , Struble Robert , Price Donald
Published: October 2006

Abstract: Radioligand binding studies were performed to characterize serotonin 5‐HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5‐HT binding, in the presence of pindolol (to block 5‐HT1A and 5‐HT1B receptors) and mesulergine (to block 5‐HTlc receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5‐HT‐labeled 5‐HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8‐hydroxydipropylaminotetralin, trifluoromethylphenylpiper‐azine, mesulergine, 4‐bromo‐2,5‐dimethoxyphenyliso‐propylamine, and ICS 205–930 had low affinity for [3H]5‐HT‐labeled 5‐HT1D sites, indicating that the pharmacology of the 5‐HT1D site is distinct from that of previously identified 5‐HT1A, 5‐HT1B, 5‐HTlc, 5‐HT2, and 5‐HT3 sites. 5‐HT1D sites in human brain have a similar pharmacology to the 5‐HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S) and guanosine 5′‐(β,γ‐imido)‐triphosphate (Gpp(NH)p), modulated the binding of [3H]5‐HT to 5‐HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5‐HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.
Heat Stress Induces Changes in Protein Synthesis and Fast Axonal Transport in Bullfrog Sensory Neurons
Authors: Stone George C. , Dougher Maureen M.
Published: October 2006

Abstract: The effects of heat stress on protein synthesis and fast axonal transport were examined in an in vitro bullfrog primary afferent neuron preparation. The magnitude of effect was determined for individual [35S]methionine‐labelled protein species separated via two‐dimensional gel electrophoresis. Elevation of temperature of the preparation from 18°C to 33 °C caused a transient inhibition of synthesis of non‐heat‐shock proteins, whereas the synthesis of a 74,000‐dalton protein increased to 927% of controls after 4 h. Similar prolonged stress conditions had no effect on the relative abundance of 36 individual, newly synthesized proteins undergoing fast axonal transport. A dramatic exception was represented by a 55,000‐dalton glycoprotein whose fast transport was increased to 291% of control. The increase in transport of this protein during a time when synthesis and transport of other non‐heat‐shock proteins were not enhanced suggests that it may play a unique role in the early cellular events that mediate survival or thermotolerance in the neuron.
Effect of Ethanol Administration on Striatal D1 and D2 Dopamine Receptors
Authors: Hruska Robert E.
Published: October 2006

Abstract: Chronic in vivo exposure of rats to ethanol in a complete liquid diet for 14 or 21 days produced a behavioral tolerance to the acute injection of ethanol. After 21 days, but not 14 days, of chronic exposure, there was a significant increase in the maximum density of striatal D1 and D2 dopamine receptors without a change in these receptors' affinities. A 24‐h withdrawal from the 21‐day exposure did not alter the observed increase in density. Both the level and duration of ethanol exposure appear to be important variables for demonstration of an increase in striatal D1 and D2 dopamine receptors.
Expression of Myelin Components in Mouse Schwann Cells in Culture
Authors: Burroni D. , White F. V. , Ceccarini C. , Matthieu J.‐M. , Costantino‐Ceccarin E.
Published: October 2006

Mouse Schwann cells cultured in vitro are capable of expressing basal levels of the major myelin components P1, P2, P0, and galactocerebroside. Numerical counts of immunostained cultures indicated that between 22 and 40% of the cells are positive up to 21 days for all of the components indicated. Electrophoretic analysis of Schwann cells labeled with a 14C‐amino acid mixture revealed the presence of proteins with relative mobilities identical to those of P0 and P1. Positive identification of the two proteins was indicated by immunoprecipitation of P1 and immunoblotting of P0. These data show that in the absence of neurites, Schwann cells in culture can express low levels of myelin characteristic components even in the absence of myelin assembly.
Rapid Determination of Norepinephrine, Dopamine, Serotonin, Their Precursor Amino Acids, and Related Metabolites in Discrete Brain Areas of Mice Within Ten Minutes by HPLC with Electrochemical Detection
Authors: Murai Shigeo , Saito Hiroko , Masuda Yoshikatsu , Itoh Tadanobu
Published: October 2006

A rapid and simple chromatographic procedure using HPLC with electrochemical detection is described for simultaneous determination of the substrates from precursor amino acids to metabolites related to synthesis and metabolism of three monoamine neurotransmitters—norepinephrine (NE), dopamine (DA), and 5‐hydroxytryptamine (5‐HT, serotonin)—in discrete brain areas of the mouse. Under the present instrumental and mobile phase conditions, the procedure permits simultaneous determination of three monoamines (NE, DA, and 5‐HT), two precursor amino acids (tyrosine and tryptophan), and four respective metabolites (3‐methoxy‐4‐hydroxyphenylglycol, 3,4‐dihydroxyphenylacetic acid, homovanillic acid, and 5‐hydroxyindoleacetic acid) within 10 min in one chromato graphic run. By varying column temperature, this procedure also permits simultaneous determination of 10–14 monoamine‐related substrates including the nine substrates described above within 15–21 min. The validity of the present procedure is demonstrated by analyzing the effect of an α2‐adrenergic agonist (clonidine) and an α2‐antagonist (yohimbine) in mouse hypothalamus.
Biochemical Defect of the hph‐1 Mouse Mutant Is a Deficiency in GTP‐Cyclohydrolase Activity
Authors: McDonald J. David , Cotton Richard G. H. , Jennings Ian , Ledley Fred D. , Woo Savio L. C. , Bode Vernon C.
Published: October 2006

A hyperphenylalaninemic mouse mutant, hph‐1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid‐dihydropteridine reductase activity is also normal. GTP‐cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP‐cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4‐dihydroxyphenylalanine (DOPA) and serotonin.
5‐Hydroxytryptamine‐Stimulated Inositol Phospholipid Hydrolysis in the Mouse Cortex Has Pharmacological Characteristics Compatible with Mediation via 5‐HT2 Receptors but This Response Does Not Reflect Altered 5‐HT2 Function After 5,7‐Dihydroxytryptamine Lesioning or Repeated Antidepressant Treatments
Authors: Godfrey P. P. , McClue S. J. , Young M. M. , Heal D. J.
Published: October 2006

5‐Hydroxytryptamine (5‐HT; 3 × 10‐8‐1 × 10‐5M) produced a dose‐dependent increase in phosphatidyl‐inositol/polyphosphoinositide (PI) turnover in mouse cortical slices, as measured by following production of 3H‐la‐belled inositol phosphates (IPs) in the presence of 10 mM LiCl. Analysis of individual IPs, in slices stimulated for 45 min, indicated substantial increases in inositol monophos‐phate (IP1; 140%) and inositol bisphosphate (IP2; 95%) contents with smaller increases in inositol trisphosphate (IP3; 51%) and inositol tetrakisphosphatc (IP4; 48%) contents. The increase in IP3 level was solely in the 1.3,4‐isomer. This response was inhibited by the nonselective 5‐HT antagonists methysergide, metergoline, and spiper‐one. It was also inhibited by the selective 5‐HT2 antagonists ketanserin and ritanserin but not by the 5‐HT1 antagonists isapirone, (‐)‐propranolol, or pindolol. 5‐HT‐stimulated IP formation was also unaltered by atropine, prazosin, and mepyramine. Lesioning brain 5‐HT neurones using 5,7‐di‐hydroxytryptamine (5.7‐DHT; 50 μg i.c.v.) produced a 210% (p < 0.01) increase in the number of 5‐HT2‐mediated head‐twitches induced by 5‐methoxy‐N,N‐dimethyltrypt‐amine (2 mg/kg). However, 5,7‐DHT lesioning had no effect on 5‐HT‐stimulated PI turnover in these mice. Similarly, an electroconvulsive shock (90 V, 1 s) given five times over a 10‐day period caused an 85% (p < 0.01) increase in head‐twitch responses but no change in 5‐HT‐stimulated PI turnover. Decreasing 5‐HT2 function by twice‐a‐day injection of 5 mg/kg of zimeldine or desipramine (DMI) produced 59% (p < 0.01) and 56% (p < 0.01), respectively, reductions in head‐twitch behaviour. Although zimeldine had no effect on 5‐HT‐stimulated IP formation, DMI treatment inhibited this by 33% at 3 μM 5‐HT (p < 0.02). Previous studies have shown an excellent correlation between head‐twitch responses and cortical 5‐HT2 receptor number in C57/B1/6 mice. Hence, it is concluded that although the 5‐HT‐stimulated PI turnover in cortical slices has the characteristics of a 5‐HT2 receptor‐mediated response, it does not, in general, reflect adaptive changes in 5‐HT2 receptor number and function.
Muscarinic Cholinergic Receptor Subtypes in Hippocampus in Human Cognitive Disorders
Authors: Smith Carthage J. , Perry Elaine K. , Perry Robert H. , Candy John M. , Johnson Mary , Bonham James R. , Dick David J. , Fairbairn Andrew , Blessed Gary , Birdsall Nigel J. M.
Published: October 2006

Total muscarinic receptor levels, the levels of the subtypes exhibiting high and low affinity for pirenzepine, and the high‐ and low‐affinity agonist states of the receptor were investigated in hippocampal tissue obtained at autopsy from mentally normal individuals and the following pathological groups: Alzheimer's disease, Parkinson's disease, Down's syndrome, alcoholic dementia, Huntington's chorea, and motor‐neurone disease. A moderate decrease in the density of both high‐affinity pirenzepine and high‐affinity agonist subtypes was found in Alzheimer's disease, whereas a trend towards an increase in the overall muscarinic receptor density was apparent in the parkinsonian patients without dementia, mainly due to an increase in the low‐affinity agonist state; the differences between the Alzheimer's disease and nondemented parkinsonian cases were highly significant. As previously reported, the levels of both choline acetyltransferase and acetylcholinesterase were markedly reduced in both Alzheimer's disease and Parkinson's disease—with a greater loss of both enzymes in the demented subgroup of parkinsonian patients. Activities of the cholinergic enzymes were also extensively reduced in Down's syndrome, accompanied by a loss of high‐affinity pirenzepine binding. There were no significant receptor or enzyme alterations in the other groups studied. These observations suggest that in the human brain, extensive degeneration of cholinergic axons to the hippocampus, as indicated by a loss of cholinergic enzymes, is not necessarily accompanied by extensive muscarinic receptor abnormalities (as might be expected if a major subpopulation were presynaptic). Moreover, the opposite changes in muscarinic binding in Parkinson's and Alzheimer's diseases may be related to the greater severity of dementia in the latter disease. Key Words: Muscarinic receptor subtypes—Human hippocampus—Alzheimer's disease—Parkinson's disease —Down's syndrome. Smith C. J. et al. Muscarinic cholinergic receptor subtypes in hippocampus in human cognitive disorders.
Brain Metabolism and Intracellular pH During Ischaemia: Effects of Systemic Glucose and Bicarbonate Administration Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy In Vivo in the Lamb
Authors: Hope P. L. , Cady E. B. , Delpy D. T. , Ives N. K. , Gardiner R. M. , Reynolds E. O. R.
Published: October 2006

Abstract: Brain metabolism and intracellular pH were studied during and after episodes of incomplete cerebral ischaemia in lambs under sodium pentobarbitone anaesthesia. 31P and 1H magnetic resonance spectroscopy was used to monitor brain pHi and brain concentrations of inorganic phosphate (Pi), phosphocreatine (PCr), β‐nucleoside triphosphate (βNTP), and lactate. Simultaneous measurements were made of arterio‐cerebral venous concentration differences (AVDs) for oxygen, glucose, and lactate. Cerebral ischaemia was induced by a combination of bilateral carotid clamping and hypotension, and the acute effects of systemic administration of glucose and sodium bicarbonate were examined. The molar ratio of glucose to oxygen uptake by the brain (6G/O2) increased above unity during cerebral ischaemia. Statistically significant AVDs for lactate were not observed. Cerebral ischaemia was associated with a reduction in brain pHi PCr/Pi ratio, and an increase in brain lactate. No effect of arterial plasma glucose on brain lactate concentration or brain pHi was evident during cerebral ischaemia or in the postischaemic period. Administration of sodium bicarbonate systemically in the postischaemic period was associated with a rise in arterial and brain tissue Pco2. A fall in brain pHi occurred which was attributable in part to coincidental brain lactate accumulation. The increase in brain lactate measured by‘H nuclear magnetic resonance in vivo during ischaemia was insufficient to account for the change in buffer base calculated to have occurred from previous estimates of brain buffering capacity.
Influence of the Malate‐Aspartate Shuttle on Oxidative Metabolism in Synaptosomes
Authors: Cheeseman Andrea J. , Clark John B.
Published: October 2006

Abstract: β‐Methyleneaspartate, a specific inhibitor of aspartate aminotransferase (EC 2.6.1.1.), was used to investigate the role of the malate‐aspartate shuttle in rat brain synaptosomes. Incubation of rat brain cytosol, “free” mitochondria, synaptosol, and synaptic mitochondria, with 2 mMβ‐methyleneaspartate resulted in inhibition of aspartate aminotransferase by 69%, 67%, 49%, and 76%, respectively. The reconstituted malate‐aspartate shuttle of “free” brain mitochondria was inhibited by a similar degree (53%). As a consequence of the inhibition of the aspartate aminotransferase, and hence the malate‐aspartate shuttle, the following changes were observed in synaptosomes: decreased glucose oxidation via the pyruvate dehydrogenase reaction and the tricarboxylic acid cycle; decreased acetylcholine synthesis; and an increase in the cytosolic redox state, as measured by the lactate/pyruvate ratio. The main reason for these changes can be attributed to decreased carbon flow through the tricarboxylic acid cycle (i.e., decreased formation of oxaloacetate), rather than as a direct consequence of changes in the NAD+/NADH ratio. Malate/glutamate oxidation in “free” mitochondria was also decreased in the presence of 2 mMβ‐methyleneaspartate. This is probably a result of decreased glutamate transport into mitochondria as a result of low levels of aspartate, which are needed for the exchange with glutamate by the energy‐dependent glutamate‐aspartate translocator.
Dopamine Receptors on Adrenal Chromaffin Cells Modulate Calcium Uptake and Catecholamine Release
Authors: Bigornia Luisa , Suozzo Margaret , Ryan Kelly A. , Napp David , Schneider Allan S.
Published: October 2006

Abstract: The presence of dopamine‐containing cells in sympathetic ganglia, i.e., small, intensely fluorescent cells, has been known for some time. However, the role of dopamine as a peripheral neurotransmitter and its mechanism of action are not well understood. Previous studies have demonstrated the presence of D2 dopamine receptors on the surface of bovine adrenal chromaffin cells using radio‐ligand binding methods and dopamine receptor inhibition of catecholamine release from perfused adrenal glands. In the present study, we provide evidence confirming a role of dopamine receptors as inhibitory modulators of adrenal catecholamine release from bovine chromaffin cell cultures and further show that the mechanism of modulation involves inhibition of stimulated calcium uptake. Apomor‐phine gave a dose‐dependent inhibition (IC50= 1 μM) of 45Ca2+ uptake stimulated by either nicotine (10 μM) or membrane depolarization with an elevated K+ level (60 mM). This inhibition was reversed by a series of specific (including stereospecific) dopamine receptor antagonists: haloperidol, spiperone, sulpiride, and (+)‐butaclamol, but not (—)‐butaclamol. In addition, the calcium channel agonist Bay K 8644 was used to stimulate uptake of 45Ca2+ into chromaffin cells, and this uptake was also inhibited by the dopamine receptor agonist apomorphine. The combined results suggest that dopamine receptors on adrenal chromaffin cells alter Ca2+ channel conductance, which, in turn, modulates catecholamine release.
Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia
Authors: Ninomiya Haruaki , Taniguchi Takashi , Kameyama Masakuni , Fujiwara Motohatsu
Published: October 2006

Abstract: We examined the effects of in vivo hypoxia (10% O2/90% N2) on the γ‐aminobutyric acid (GABA)/benzo‐diazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6‐h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X‐100 eliminated the increase in the binding capacity. These results may represent an up‐regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.
Purifications of Rat Adrenal Dopamine‐β‐Hydroxylase: Immunological Analysis of Its Soluble and Membrane‐Bound Forms with the Use of an Antibody Raised Against the Soluble Form
Authors: Richard Francoise , Buda Michel , Legay Francois
Published: October 2006

Abstract: Soluble and membrane‐bound dopamine‐β‐hy‐droxylases (sDBH and mDBH, respectively) from rat adrenal glands have been purified through concanavalin A‐Sepharose chromatography, gel filtration, and ion‐exchange high‐performance chromatographies. Both sDBH and mDBH were composed by four subunits of apparent molecular weight of 57,000 and showed a native molecular weight of 300,000. This procedure has not allowed us to obtain a sufficient amount of enzyme to immunize a rabbit. A second, more rapid procedure was designed to isolate sDBH, including concanavalin A‐Sepharose chromatography and preparative sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A rabbit antiserum was raised against this purified protein. The specificity of the antiserum was demonstrated by neutralization of rat adrenal gland DBH activity, labeling of rat adrenal medulla on histological sections, and, after Western blot, labeling of the 75,000–molecular‐weight band in the different fractions associated with DBH activity during purification. The antiserum had a higher affinity for the sDBH denatured by sodium dodecyl sulfate than for the native protein. It had a higher affinity for sDBH than for mDBH. These results strongly suggest the presence of specific hydrophilic epitopes on the sDBH, revealing structural differences between the two hydroxylase forms. Two protein bands were stained on Western blots of crude rat adrenal gland extract. One band had an apparent molecular weight of 75,000, and the other of 82,000. Our results showed that the two proteins contained similar epitopes, an observation suggesting a close structural relationship. The higher‐molecular‐weight protein could be the75,000 protein before covalent modifications and cleavage.