bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Oestradiol and Progesterone on Hypoglycaemic Stress‐Induced Suppression of Pulsatile Luteinizing Hormone Release and on Corticotropin‐Releasing Hormone mRNA Expression in the Rat
Authors: Li X. F. , Mitchell J. C. , Wood S. , Coen C. W. , Lightman S. L. , O'Byrne K. T.
Published: July 2010

Corticotropin‐releasing hormone (CRH) is implicated in the suppression of pulsatile luteinizing hormone (LH) secretion by a variety of stressful stimuli; 17β‐oestradiol (E2) has been shown to modulate this inhibitory response. The present study in ovariectomized (OVX) rats was designed to investigate the effect of E2 and progesterone (P4) on hypoglycaemic stress‐induced changes in pulsatile LH secretion and on the associated changes in both central and peripheral components of the hypothalamic‐pituitary‐adrenal axis. E2 enhanced the hypoglycaemic stress‐induced suppression of LH pulses; P4 in addition to E2 further potentiated the inhibitory response. The rise in plasma corticosterone following insulin‐induced hypoglycaemia (IIH) was highest in the E2 + P4 group. Nevertheless, when such levels were achieved by administration of corticosterone, the occurrence of LH pulses was completely unaffected, irrespective of ovarian steroid milieu. E2 and E2 + P4 up‐regulated basal CRH mRNA expression in the paraventricular nucleus (PVN) as measured by in situ hybridization; this signal was also increased in the medial preoptic nucleus (MPN) following E2. IIH resulted in a rise in CRH mRNA in the PVN, but not in the MPN; this rise may reflect a more significant role for the PVN in the present context. Changes in neuropeptide mRNA expression may signal changes in neuronal activity; nevertheless, the profound differences in LH pulse suppression in OVX, E2 and E2 + P4 rats following IIH were not reflected in the concurrent changes in CRH mRNA in the PVN. The results suggest that while corticosterone has no acute effect on LH pulses in the rat, the up‐regulation by ovarian steroids of basal CRH mRNA in the PVN and/or MPN may contribute to the central regulation of these pulses in response to stress.
Corticotropin‐Releasing Hormone (CRH) Expression and Protein Kinase A Mediated CRH Receptor Signalling in an Immortalized Hypothalamic Cell Line
Authors: Kasckow J. , Mulchahey J. J. , Aguilera G. , Pisarska M. , Nikodemova M. , Chen H.‐C. , Herman J. P. , Murphy E. K. , Liu Y. , Rizvi T. A. , Dautzenberg F. M. , Sheriff S.
Published: July 2010

Corticotropin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH‐expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus‐mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH‐like immunoreactivity (CRH‐LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase‐polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule‐associated protein‐2. We verified that the CRH‐LI from IVB cell lysates coeluted with CRH standard via reversed‐phase high‐performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration‐dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho‐cyclic‐AMP response element binding protein (CREB) imunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB‐mediated gene expression as determined via the PathDetect CREB trans‐reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH‐R1‐mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.
Sexual Differentiation of the Neuroendocrine Mechanisms Regulating Mate Recognition in Mammals
Authors: Bakker J.
Published: July 2010

When in breeding condition, male and female mammals seek out and mate with opposite‐sex conspecifics. The neural mechanisms controlling mate recognition and heterosexual partner preference are sexually differentiated by the perinatal actions of sex steroid hormones. Many mammalian species use odours to identify potential mates. Thus, sex differences in partner preference may actually reflect sex differences in how male and female mammals perceive socially relevant odours. Two olfactory systems have evolved in vertebrates that differ considerably in their anatomy and function. It is generally believed that the main olfactory system is used to detect a wide variety of volatile odours derived from food prey among many sources, whereas the accessory olfactory system has evolved to detect and process primarily nonvolatile odours shown to influence reproductive behaviours and neuroendocrine functions. Some recent results obtained in oestradiol‐deficient aromatase knockout (ArKO) mice that provide evidence for a developmental role of oestradiol in olfactory investigation of volatile body odours are discussed, suggesting that: (i) oestrogens contribute to the development of the main olfactory system and (ii) mate recognition is mediated by the main as opposed to the accessory olfactory system. Thus, sex differences in mate recognition and sexual partner preference may reflect sex differences in the perception of odours by the main olfactory system.
Quantitative Analysis of Corticotropin‐Releasing Factor and Arginine Vasopressin mRNA in the Hypothalamus During Chronic Morphine Treatment in Rats: AnIn SituHybridization Study
Authors: Laorden M. L. , Milanés M. V. , Angel E. , Tankosic P. , Burlet A.
Published: July 2010

The content of corticotropin‐releasing factor (CRF) and arginine vasopressin (AVP) in the hypothalamic paraventricular nucleus (PVN) increases during chronic morphine treatment. Because these experiments cannot distinguish between increased synthesis or reduced release, the present study measured changes in CRF and AVP mRNAs in the PVN byin situhybridization. Concomitantly, changes in noradrenaline turnover in the PVN and changes in plasma corticosterone release were determined. Male rats were implanted with placebo (naive) or morphine pellets for 7 days. On day 7, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg, i.p.). Acute morphine injection did not change the total size of the labelled area for CRF mRNA in the PVN of naive or morphine‐pelleted rats, indicating that the number of CRF‐containing neurones was unchanged. On the other hand, in rats chronically treated with morphine, the intensity of labelling for CRF mRNA was significantly reduced, suggesting a decrease in the synthesis of CRF. In placebo rats, injection of saline or morphine did not affect the surface hybridized for AVP mRNA. By contrast, in the morphine‐group injected with saline, there was a significant reduction in the number of labelled neurones, measured by the size of labelled area. Similarly, there was a decrease in intensity of AVP mRNA expression in the parvocellular and magnocellular neurones of the PVN in the morphine‐group injected with saline, suggesting a decreased synthesis of AVP in these neurones. In parallel with the decrease in the expression of CRF and AVP mRNAs in the PVN, there was a pronounced decrease in noradrenaline turnover and in the release of corticosterone in the morphine‐pelleted rats. In conclusion, present results show that, in addition to modifications in corticosterone secretion and in noradrenaline turnover, chronic morphine administration produces a reduction in the synthesis of CRF and AVP.
Neuropeptide Y Targets in the Hypothalamus: Nitric Oxide Synthesizing Neurones Express Y1 Receptor
Authors: Fetissov S. O. , Xu Z. ‐Q. , Byrne L. C. , Hassani H. , Ernfors P. , Hökfelt T.
Published: July 2010

Neuropeptide Y (NPY)‐expressing neurones in the arcuate nucleus densely innervate many hypothalamic nuclei. To determine the neurochemical phenotype of target neurones for NPY, we studied the immunohistochemical localization of the NPY Y1 receptor (Y1R) in discrete subpopulations of neurones in the rat hypothalamus. Among several tested populations, including hypocretin/orexin‐, melanin‐concentrating hormone (MCH)‐ and nitric oxide synthase (NOS)‐positive neurones, only the latter were found to coexpress the Y1R. Numerous Y1R/NOS‐positive neurones were found as a densely packaged group of cells located ventrolateral to the ventromedial nucleus, forming a band ascending towards the fornix. Lower numbers of Y1R/NOS‐positive neurones were found in the perifornical area and in the peri‐ and paraventricular nuclei. Expression of the Y1R gene was found in the same locations in the mouse by colocalizing β‐galactosidase, a Y1R gene reporter, with NOS in a Y1R knockout mouse. To explore possible downstream targets of NO in the rat hypothalamus, the NO‐regulated molecule cGMP was analysed immunohistochemically after incubation of brain slices with sodium nitroprusside, an NO donor. We observed several cGMP‐positive cell bodies in the arcuate nucleus, cGMP‐positive blood vessels and a cGMP‐positive network of thin fibres, some of which colocalized with choline acetyltransferase.
Early Social Stress in Male Guinea‐Pigs Changes Social Behaviour, and Autonomic and Neuroendocrine Functions
Authors: Kaiser S. , Kruijver F. P. M. , Straub R. H. , Sachser N. , Swaab D. F.
Published: July 2010

The present study was undertaken to investigate the effects of pre‐ and early postnatal social stress on the functioning of hormonal, autonomic and behavioural systems, by studying the distribution of sex hormone receptors in limbic brain systems. Dams had either lived in groups with a constant composition (= stable social environment) or in groups with changing compositions, i.e. every third day, two females from different groups were exchanged (= unstable social environment). The subjects were male offspring of dams who had either lived in a stable social environment during pregnancy and lactation (= control males) or in an unstable social environment during this period of life (= early stressed males). From days 20–80, the spontaneous behaviour of control males and early stressed males was recorded in their home cages. Five control males and five early stressed males were killed at 75 days, and five control males and five early stressed males at 120 days. Blood samples were taken to determine serum concentrations of cortisol, testosterone, dehydroepiandrosterone, dehydroepiandrosterone sulphate and oestrogen. The adrenals were prepared to determine tyrosine hydroxylase activities and the brains were used to investigate the distribution of sex‐hormone receptors in specific hypothalamic and hippocampal brain areas. Early stressed males showed a behavioural infantilization that was accompanied by significantly decreased adrenal tyrosine hydroxylase activities and dehydroepiandrosterone levels. Furthermore, early stressed males showed a down‐regulation of androgen receptors in the medial preoptic area and the nucleus arcuatus of the hypothalamus, as well as of oestrogen receptor α in the hippocampus compared to control males. Thus, the present study provides clear evidence that early social stress induces changes in endocrine, autonomic and limbic brain function, which is mirrored by changes in male social behaviour.
Studies on the Neuroanatomical Basis for Stress‐Induced Oestrogen‐Potentiated Suppression of Reproductive Function: Evidence Against Direct Corticotropin‐Releasing Hormone Projections to the Vicinity of Luteinizing Hormone‐Releasing Hormone Cell Bodies in Female Rats
Authors: Hahn J. D. , Kalamatianos T. , Coen C. W.
Published: July 2010

Various studies implicate corticotropin‐releasing hormone (CRH) as a mediator for the inhibitory effects of stress on reproduction. This study was designed to elucidate the underlying neuroanatomy. The retrograde tracer cholera toxin was picospritzed into the vicinity of the luteinizing hormone‐releasing hormone (LHRH) perikarya. CRH neurones were examined for the tracer in the medial preoptic nucleus (MPO), bed nucleus of the stria terminalis (BST), paraventricular nucleus (PVN), central amygdaloid nucleus (CeM), parabrachial nucleus (PB) and additional locations. Retrograde label was not detected in CRH neurones at any of these sites; nevertheless, in the MPO and PB, abundant retrogradely‐labelled perikarya intermingled with CRH neurones. In the BST, CeM and PVN, sites containing major CRH cell populations, retrogradely‐labelled cells were scarce or absent; however, retrograde labelling was found in adjacent regions: lateral septum, medial amygdaloid nucleus and areas bordering the PVN. Double‐label in situ hybridization for the mRNAs for LHRH and the CRH type‐1 receptor (CRH‐R1) identified the receptor transcript at sites rostral and lateral to the LHRH neurones (in the vertical and horizontal limbs of the diagonal band) but not in the LHRH neurones. Given the ability of oestrogen to potentiate stress‐induced suppression of LH release, the identification of CRH neurones immunoreactive for oestrogen receptor (ER)α in the MPO and for ERβ in the caudal PVN may be significant. In this context, it is also noteworthy that CRH neurones within the MPO and PB which are, respectively, immunopositive and immunonegative for ERα, lie within the vicinity of retrogradely‐labelled cells. The present findings suggest that the means by which CRH may mediate inhibitory effects of stressors on LH release do not involve direct CRH projections to LHRH neurones; the indirect means for such regulation, and the sites at which oestrogen may potentiate the inhibitory response, remain to be established.
Gonadotropin‐Inhibitory Peptide in Song Sparrows (Melospiza melodia) in Different Reproductive Conditions, and in House Sparrows (Passer domesticus) Relative to Chicken‐Gonadotropin‐Releasing Hormone
Authors: Bentley G. E. , Perfito N. , Ukena K. , Tsutsui K. , Wingfield J. C.
Published: July 2010

Gonadotropin‐releasing hormone (GnRH) regulates reproduction in all vertebrates. Until recently, an antagonistic neuropeptide for gonadotropin was unknown. The discovery of an RFamide peptide in quail that inhibits gonadotropin release in vitro raised the possibility of direct hypothalamic inhibition of gonadotropin release. This peptide has now been named gonadotropin‐inhibitory hormone (GnIH). We investigated GnIH presence in the hypothalamus of two seasonally breeding songbird species, house sparrows (Passer domesticus) and song sparrows (Melospiza melodia). Using immunocytochemistry (ICC), GnIH‐containing neurones were localized in both species in the paraventricular nucleus, with GnIH‐containing fibres visible in multiple brain locations, including the median eminence and brainstem. Double‐label ICC with light microscopy and fluorescent ICC with confocal microscopy indicate a high probability of colocalization of GnIH with GnRH neurones and fibres within the avian brain. It is plausible that GnIH could be acting at the level of the hypothalamus to regulate gonadotropin release as well as at the pituitary gland. In a photoperiod manipulation experiment, GnIH‐containing neurones were larger in birds at the termination of the breeding season than at other times, consistent with a role for this neuropeptide in the regulation of seasonal breeding. We have yet to elucidate the dynamics of GnIH synthesis and release at different times of year, but the data imply temporal regulation of this peptide. In summary, GnIH has the potential to regulate gonadotropin release at more than one level, and its distribution is suggestive of multiple regulatory functions in the central nervous system.
Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit
Authors: Kamboj Rajender K. , Schoepp Darryle D. , Nutt Stephen , Shekter Lee , Korczak Bozena , True Rebecca A. , Rampersad Vikarna , Zimmerman Dennis M. , Wosnick Michael A.
Published: July 2010

Abstract:Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high‐affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [3H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L‐glutamate > (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid > dihydro‐kainate > 6, 7‐dinitroquinoxaline‐2, 3‐dione > 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand‐gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high‐affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.
Endopeptidases 24.16 and 24.15 Are Responsible for the Degradation of Somatostatin, Neurotensin, and Other Neuropeptides by Cultivated Rat Cortical Astrocytes
Authors: Mentlein Rolf , Dahms Peter
Published: July 2010

Abstract: Several neuropeptides, including neurotensin, somatostatin, bradykinin, angiotensin II, substance P, and luteinizing hormone‐releasing hormone but not vasopressin and oxytocin, were actively metabolized through proteolytic degradation by cultivated astrocytes obtained from rat cerebral cortex. Because phenanthroline was an effective degradation inhibitor, metalloproteases were responsible for neuropeptide fragmentation. Neurotensin was cleaved by astrocytes at the Pro10‐Tyr11 and Arg8‐ Arg9 bonds, whereas somatostatin was cleaved at the Phe6‐Phe7 and Thr10‐Phe11 bonds. These cleavage sites have been found previously with endopeptidases 24.16 and 24.15 purified from rat brain. Addition of specific inhibitors of these proteases, the dipeptide Pro‐He and N‐[1‐(RS)‐carboxy‐3‐phenylpropyl]‐Ala‐Ala‐Phe‐4‐aminobenzoate, significantly reduced the generation of the above neuropeptide fragments by astrocytes. The presence of endopeptidases 24.16 and 24.15 in homogenates of astrocytes could also be demonstrated by chromatographic separations of supernatant solubilized cell preparations. Proteolytic activity for neurotensin eluted after both gel and hydroxyapatite chromatography at the same positions as found for purified endopeptidase 24.16 or 24.15. In incubation experiments or in chromatographic separations no phosphoramidon‐sensitive endopeptidase 24.11 (enkephalinase) or captopril‐sensitive peptidyl dipeptidase A (angiotensin‐converting enzyme) could be detected in cultivated astrocytes. Because astrocytes embrace the neuronal synapses where neuropeptides are released, we presume that the endopeptidases 24.16 and 24.15 on astrocytes are strategically located to contribute significantly to the inactivation of neurotensin, somatostatin, and other neuropeptides in the brain.
On the Origin of Striatal Cholecystokinin Release: Studies with In Vivo Microdialysis
Authors: You Z.‐B. , Herrera‐Marschitz M. , Brodin E. , Meana J. J. , Morino P. , Hokfelt T. , Silveira R. , Goiny M. , Ungersfedt U.
Published: July 2010

Abstract: In the present study, extracellular levels of the neuropeptide cholecystokinin (CCK), of the monoamine dopamine and its metabolites 3, 4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and of the excitatory amino acids glutamate and aspartate were simultaneously monitored by microdialysis in the neostriatum of halothane‐anesthetized rats under basal and K+‐depolarizing conditions. Extracellular CCK and dopamine levels, but not glutamate and aspartate levels, were decreased by perfusion with a Ca2+‐free medium, under both basal and K+‐depolarizing conditions. HPLC revealed that the majority of the CCK‐like immunoreactivity in the perfusates coeluted with CCK octapeptide. Striatal extracellular CCK levels were decreased by decortication plus callosotomy, with a parallel decrease in glutamate levels. Striatal extracellular levels of dopamine, DOPAC., and HVA were significantly decreased in animals treated previously with a unilateral 6‐hydroxydopamine injection into the medial forebrain bundle. In these animals, however, the effect of decortication plus callosotomy on CCK and glutamate levels was not further augmented. Thus, this study supports the hypothesis of a neuronal origin of extracellular CCK and dopamine monitored with microdialysis in the striatum of the rat, and also supports the idea of a partly contralateral origin of corticostriatal CCK and glutamate inputs.
Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on theN‐ Methyl‐D‐Aspartate Receptor lonophore Complex in Rat Brain
Authors: Yoneda Yukio , Suzuki Takeo , Ogita Kiyokazu
Published: July 2010

Abstract:Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [3H] 5, 7‐dichlorokynurenic acid ([3H]‐ DCKA) but not of the agonist ligand [3H] glycine ([3H] Gly) to a Gly recognition domain on theN‐methyl‐D‐aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [3H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)‐α‐(4‐chlorophenyl)‐4‐ [(4‐fluorophenyl)methyl]‐1‐piperidine ethanol, with [3H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [3H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A2and C andp‐chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [3H] DCKA binding with [3H] Gly binding being unaltered. Moreover, the densities of [3H] DCKA binding were not significantly different from those of [3H]‐ Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [3H] Gly binding than of [3H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.
Increased Iron in the Substantia Nigra Compacta of the MPTP‐Lesioned Hemiparkinsonian African Green Monkey: Evidence from Proton Microprobe Elemental Microanalysis
Authors: Temlett J. A. , Landsberg J. P. , Watt F. , Orime G. W.
Published: July 2010

Abstract:The association of free radicals and particularly free iron in the pathogenesis of idiopathic Parkinson's disease and MPTP‐induced parkinsonism remains controversial. Whereas the actual cause of dopamine cell death in the substantia nigra compacta (SNc) remains unknown, disturbances in lipid peroxidation and subsequent mitochondrial and cell membrane disruption has been demonstrated. In a genetically susceptible host, abnormal elimination of oxygen and trace metal free radicals may further damage dopamine cells. Using a unilaterally MPTP‐treated African Green monkey, which showed obvious contralateral hemiparkinsonism, the total free iron concentration was measured. Iron, Fe2+and Fe3+, but not other trace elements, was significantly elevated in the SNc compared with the opposite unlesioned side, which was similar to separate control animals. Iron content in the SNc, periaqueductal gray area, and crus cerebri was 228–270 ppm. Normal control SNc was 285 (±59) ppm, whereas iron levels of 532 (±151) ppm were found in the MPTP‐lesioned SNc. These animals were drug naive and not on long‐term levodopa maintenance. Proton microprobe elemental analysis was matched against adjacent immunocytochemically stained tissue slices to ensure the cells studied were in the SNc. Iron was found not only in the degenerating dopamine cells themselves but also in the surrounding matrix and glial cells. Whether free iron that is not bound to neuromelanin is responsible for dopamine cell death as suggested by these experiments remains to be proved.
Decreased myo‐lnositol Uptake Is Associated with Reduced Bradykinin‐Stimulated Phosphatidylinositol Synthesis and Diacylglycerol Content in Cultured Neuroblastoma Cells Exposed to L‐Fucose
Authors: Yorek Mark A. , Dunlap Joyce A. , Stefani Mark R. , Davidson Eric P. , Zhu Xi , Eichberg Joseph
Published: July 2010

Abstract: L‐Fucose is a potent, competitive inhibitor of myo‐inositol transport by cultured mammalian cells. Chronic exposure of neuroblastoma cells to L‐fucose causes a concentration‐dependent decrease in myo‐inositol content, accumulation, and incorporation into phosphoinositides. In these studies, L‐fucose supplementation of culture medium was used to assess the effect of decreased myo‐inositol metabolism and content on bradykinin‐stimulated phosphatidylinositol synthesis and diacylglycerol production. Chronic exposure of cells to 30 mML‐fucose caused a sustained decrease in bradykinin‐stimulated, but not basal, 3H‐inositol phosphate release and 32P incorporation into phosphatidylinositol in cells incubated in serum‐free, unsupplemented medium. In addition, 32P incorporation into phosphatidylinositol 4‐phosphate and phosphatidylinositol 4, 5‐bisphosphate was not altered in L‐fucose‐conditioned cells. Acute exposure of cells to serum‐free medium containing 30 mM L‐fucose did not affect either basal or bradykinin‐stimulated 32P incorporation into phosphatidylinositol. Basal diacylglycerol content was decreased by 20% in cells chronically exposed to 30 mM L‐fucose, although analysis of the molecular species profile revealed no compositional change. Bradykinin stimulated diacylglycerol production in neuroblastoma cells by increasing the hydrolysis of both phosphoinositides and phosphatidylcholine. Bradykinin‐stimulated production of total diacylglycerol was similar for control and L‐fucose‐conditioned cells. However, there was a decrease in the bradykinin‐induced generation of the 1 ‐stearoyl‐2‐arachidonoyl diacylglycerol molecular species in the cells chronically exposed to 30 mM L‐fucose. This molecular species accounts for about 70% of the composition of phosphoinositides, but only 10% of phosphatidylcholine. The results suggest that a decrease in myo‐inositol uptake results in diminished agonist‐induced phosphatidylinositol synthesis and phosphoinositide hydrolysis in cultured neuroblastoma cells grown in L‐fucose‐containing medium.
Agonist‐Induced, GTP‐Dependent Phosphoinositide Hydrolysis in Postmortem Human Brain Membranes
Authors: Jope Richard S. , Song Ling , Powers Richard
Published: July 2010

Abstract:Membranes prepared from postmortem human brain were used to measure the activities of three components of the phosphoinositide second messenger system. [3H] Phosphatidylinositol ([3H] PI) hydrolysis was stimulated by directly activating phospholipase C with calcium, by activating guanine nucleotide‐binding proteins (G proteins) with guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) or with AIF4, and by receptors activated with several agonists (in the presence of GTPγS), including (in order of increasing magnitudes of responses) carbachol, pilocarpine, histamine,trans‐1‐aminocyclopentyl‐1, 3‐dicarboxylic acid (a selective excitatory amino acid metabotropic receptor agonist), serotonin, and ATP. Gq/11was identified as the G protein most likely to mediate [3H] PI hydrolysis in human brain membranes based on the findings that this process was not impaired by pretreatment with pertussis toxin and it was inhibited by antibodies specific for the α‐subunit of Gq/11but not by antibodies for Goor G11. The effects of postmortem delay on [3H] PI hydrolysis were examined by studying tissues obtained 6–21 h postmortem. A slight increase in basal [3H] PI hydrolysis was associated with increased postmortem time, suggesting a slow loss of the normal inhibitory control of phospholipase C. GTPγS‐ stimulated [3H] PI hydrolysis was unaffected by postmortem times within this range, but carbachol‐induced [3H] PI hydrolysis tended to decrease with increasing postmortem times. These results demonstrate that the entire phosphoinositide complex remains functional and experimentally detectable in postmortem human brain membranes. This method provides a means to study the function, regulation, effects of diseases, and responses to drugs of the phosphoinositide system in human brain.
Selective Lesions by Manganese and Extensive Damage by Iron After Injection into Rat Striatum or Hippocampus
Authors: Sloot W. N. , Sluijs‐Gelling A. J. van der , Grambbergen J. B. P.
Published: July 2010

Abstract:Regional45Ca2+accumulation and analysis of monoamines and metabolites in dissected tissues were used to localize, quantify, and characterize brain damage after intracerebral injections of Mn2+into striatum and hippocampus. The specificity of Mn2+‐induced lesions is described in relation to brain damage produced by local Fe2+or 6‐hydroxydopamine (6‐OHDA) injections. In striatum, Fe2+and Mn2+produced dose‐dependent (0.05‐0.8 μmol) dopamine (DA) depletion, with Fe2+being 3.4 times more potent than Mn2+. Studies examining the time course of changes in monoamine levels in striatum following local application of 0.4 μmol of Mn2+revealed maximal depletion of all substances investigated (except 5‐hydroxyin‐doleacetic acid) after 3 days. The effects on DA (87% depletion at day 3) and its major metabolites were most pronounced and lasted until at least 90 days (40% depletion), whereas serotonin and noradrenaline levels recovered within 21 and 42 days, respectively. In addition, levels of 3‐methoxytyramine, which is used as an index of DA release, also recovered within 42 days, indicating a functional restoration of DA neurotransmission despite substantial loss of DA content. Intrastriatal Mn2+(0.4 μmol) produced time‐dependent45Ca2+accumulation in striatum, globus pallidus, entopeduncular nucleus, several thalamic nuclei, and substantia nigra pars reticulata ipsilateral to the injection site. In contrast, 6‐OHDA injected at a dose equipotent in depleting DA produced significantly less45Ca2+accumulation in striatum and globus pallidus and no labeling of other brain areas, whereas Fe2+(0.4 μmol) produced extensive45Ca2+accumulation throughout basal ganglia, accumbens, and cerebral cortex. In hippocampus, high Mn2+(0.4 μmol) produced limited45Ca2+accumulation in subiculum and dentate gyrus, whereas low Fe2+(0.1 μmol) produced widespread45Ca2+accumulation throughout hippocampus, thalamus, and cerebral cortex. It is concluded that (a) Mn2+is selectively neurotoxic to pathways intrinsic to the basal ganglia, (b) intrastriatal injections can be used as a model for systemic Mn2+intoxications, and (c) high endogenous Fe3+and/or catecholamine levels potentiate the neurotoxicity of Mn2+.
Contrasting Effects of D‐ and L‐(E)‐4‐(3‐Phospiono‐2‐ Propenyl) Piperazine‐2‐Carboxylic Acid as Anticonvulsants and as Inhibitors of Potassium‐Evoked Increases in Hippocampal Extracellular Glutamate and Aspartate Levels in Freely Moving Rats I
Authors: Millan M. H. , Chapman A. G. , Meldrum B. S.
Published: July 2010

Abstract:Microdialysis experiments performed in the dorsal hippocampus of freely moving rats showed that L‐(E)‐ 4‐(3‐phosphono‐2‐propenyl) piperazine‐2‐carboxylic acid (L‐CPPene) is 10 times as potent as D‐CPPene in inhibiting potassium‐induced increases in extracellular levels of aspartate and glutamate. In control experiments, two 100 mMKCI stimuli (S1 and S2) applied for 10 min each (separated by a 40‐min recovery period) produced substantial (300–500%) increases in the extracellular levels of aspartate, glutamate, taurine, and GABA and a 50% decrease in the glutamine level. S2/S1 ratios in the control groups were 0.67 (aspartate), 0.78 (glutamate), 0.83 (GABA), and 0.85 (taurine). In the experimental groups, D‐ or L‐CPPene was applied via the probe during the second potassium stimulus (S2). L‐CPPene (25 or 250 μM) produced selective suppression of potassium‐induced increases of extracellular glutamate (S2/S1 ratio: 0.25) and aspartate (S2/S1 ratio: 0.20) levels, whereas 250 μMD‐CPPene was required to inhibit the extracellular aspartate and glutamate increases. Neither enantiomer of CPPene affected the potassium‐induced increases of GABA and taurine or the decrease in extracellular glutamine concentration. An addtional study comparing the anticonvulsant potencies of D‐ and L‐CPPene was performed using audiogenic DBA/2 mice. The anticonvulsant potency of D‐CPPene, as assessed against sound‐induced seizures in DBA/2 mice, was an order of magnitude higher than that of L‐CPPene [ED50clonic phase (intraperitoneal, 45 min): 1.64 μmol/kg and 16.8 μmol/kg, respectively]. We attribute the anticonvulsant action of D‐CPPene to its antagonist action at the NMDA receptor. The selective inhibition by L‐CPPene of potassium‐induced increases in extracellular aspartate and glutamate levels is presumably due to an action on presynaptic glutamate receptors.
Differences in the Distribution of Cytochrome b561 and Synaptophysin in Dog Splenic Nerve: A Biochemical and Immunocytochemical Study
Authors: Annaert W. G. , Quatacker J. , Llona I. , De Potter W. P.
Published: July 2010

Abstract: Compared with neurons of the CNS, the organization of the peripheral adrenergic axon and nerve terminal is more complex because two types of neurotrarismitter‐containing vesicles, i.e., large (LDVs) and small densecore vesicles, coexist with the axonal reticulum (AR) and the well‐characterized small synaptic vesicles. The AR, which is still poorly examined, is assumed to play some role in neurosecretion. We have studied the subcellular localization of noradrenaline, cytochrome b561, and synaptophysin in control and ligated dog splenic nerve using both biochemical and ultrastructural approaches. Noradrenaline and cytochrome b561 coaccumulated proximal to a ligation, whereas distally only the latter was found. Despite a codistribution with noradrenaline at high densities in sucrose gradients, Synaptophysin did not accumulate on either side of the ligation. At the ultrastructural level, cytochrome b561 immunoreactivity was found on LDVs and AR elements, both accumulating proximal to the ligation. Distally, the multivesicular bodies (MVBs), immunolabeled for cytochrome b561, account for the retrograde transport of LDVs and AR membranes retrieved at the nerve terminal. No Synaptophysin immunoreactivity could be detected on LDVs, AR, or MVBs. The results obtained from the ligation experiments together with the ultrastructural data Clearly illustrate that Synaptophysin is absent from LDVs and AR elements in adrenergic axons.
[3H] GBR‐12935 Binding to Human Cerebral Cortex Is Not to Dopamine Uptake Sites
Authors: Allard Per , Danielsson Margareta , Papworth Karin , Marousson Jan O.
Published: July 2010

Abstract:The binding of the dopamine uptake inhibitor [3H] GBR‐12935 to 16 regions of the human brain was investigated in competition experiments with increasing concentrations of GBR‐12909, mazindol, and dopamine. The methodology used included a relatively high tissue concentration (8 mg/ml) and addition of 5 mMKCI in the assay buffer. GBR‐12909 inhibited 80–90% of the binding in most regions, whereas dopamine only inhibited the binding in the striatum. Mazindol inhibited only part of the cortical binding at concentrations of >1 μM, whereas the inhibition in the caudate and the putamen also contained a high‐affinity component representing the dopamine uptake site. It is concluded that the [3H] GBR‐12935 binding sensitive to GBR‐12909 cannot be regarded as specific binding to the dopamine uptake site because the displaceable binding most likely is not related to the dopamine uptake site.
[3H] GBR‐12935 Binding to Cytochrome P450 in the Human Brain
Authors: Allard Per , Marcusson Jan O. , Ross Svante B.
Published: July 2010

Abstract:The presence of multiple [3H] GBR‐12935 binding sites in the human brain has been revealed in several recent studies. One site represents the dopamine uptake site. In rat brain it was demonstrated that [3H] GBR‐12935 also binds to nondopaminergic “piperazine acceptor sites.” One of these sites has been identified as cytochrome P450IID1 in canine brain. [3H] GBR‐12935 binding to the piperazine acceptor sites in the human brain was investigated in the present study. A pharmacological definition of the piperazine acceptor sites is presented: the [3H]‐ GBR‐12935 binding fraction that could be discriminated by 10 μMGBR‐12909 in the presence of 0.3 μMmazindol. This binding fraction was saturable, with binding affinity in the range of 3–8 nM.It was also demonstrated that the piperazine acceptor or cytochrome P450‐sensitive drugscis‐flupentixol and proadifen (SKF 525 A) compete for the same binding sites, suggesting the cytochrome P450 nature of the binding. The findings presented support the proposal that at least part of this fraction represents cytochrome P450IID6, the human form of P450IID1. The distribution of [3H] GBR‐12935 binding to the suggested P450IID6‐site in 12 brain regions was examined, without significant differences in binding densities between the regions. The significance of the present findings on the cytochrome P450 system in brain is discussed.
Evidence for a Glycidic‐Lipidic Matrix in Human Neuromelanin, Potentially Responsible for the Enhanced Iron Sequestering Ability of Substantia Nigra
Authors: Aime S. , Fasano M. , Bergamasco B. , Lopiano L. , Valente G.
Published: July 2010

Abstract: The high‐resolution solid‐state 13C‐NMR spectrum of a neuromelanin specimen (from patients dying from nonneurological diseases) is compared with that obtained from enzymatically prepared dopamine‐melanin. The main differences between the two spectra suggest the occurrence in neuromelanin of a glycidic/lipidic matrix tightly associated with the melanin macromolecule. Atomic emission spectroscopy revealed high iron content (1.5%) in the neuromelanin specimen, in full agreement with previous reports. These observations support the view that neuromelanin acts as a strong chelating (and insolubilizing) system for iron ions and further suggest that the attack to this compact composite substrate may be an important step to allow the release of iron ions responsible for the increased lipid peroxidation reported in the pathogenesis of Parkinson's disease.
Rapid Communication: Ca2+ Channel Blockers Attenuate β‐Amyloid Peptide Toxicity to Cortical Neurons in Culture
Authors: Weiss John H. , Pike Christian J. , Cotman Carl W.
Published: July 2010

Abstract: Deposit of β‐amyloid protein (Aβ) in Alzheimer's disease brain may contribute to the associated neurodegeneration. We have studied the neurotoxicity of Aβ in primary cultures of murine cortical neurons, with the aim of identifying pharmacologic ways of attenuating the injury. Exposure of cultures to Aβ (25–35 fragment; 3–25 4mUM) generally triggers slow, concentration‐dependent neurodegeneration (over 24–72 h). With submaximal Aβ‐ (25–35) exposure (10 μM), substantial (>40% within 48 h) degeneration often occurs and is markedly attenuated by the presence of the Ca2+ channel blockers nimodipine (1–20 μM) and Co2+ (100 μM) during the Aβ exposure. However, Aβ neurotoxicity is not affected by the presence of glutamate receptor antagonists. We suggest that Ca2+ influx through voltage‐gated Ca2+ channels may contribute to Aβ‐induced neuronal injury and that nimodipine and Co2+, by attenuating such influx, are able to attenuate Aβ neurotoxicity.
Rapid Communication: Nerve Growth Factor Increases the Transcriptional Activity of the Rat Neuronal Nicotinic Acetylcholine Receptor β4 Subunit Promoter in Transfected PC12 Cells
Authors: Hu Minjie , Whiting Theobald Narda L. , Gardner Paul D.
Published: July 2010

Abstract: Neuronal nicotinic acetylcholine receptors play a key role in synaptic transmission in the nervous system. Although complementary DNA clones encoding a family of acetylcholine receptor subunits have been isolated and subsequent anatomical studies indicate differences in the temporal and spatially restricted patterns of expression of each gene, the cellular and molecular mechanisms controlling the expression of these genes are unknown. As part of a long‐term goal to elucidate these mechanisms, we have been identifying and characterizing regions of the receptor subunit genes involved in transcriptional regulation. Here, we report the localization of the transcription initiation site of the rat β4 subunit gene, demonstrate using transient transfection analysis of PC12 cells that sequences upstream of this site are capable of activating transcription of a heterologous gene, and show that this transcriptional activity is enhanced in PC12 cells by treatment with nerve growth factor.
Regulation of β‐Adrenergic Receptor mRNA in Rat C6 Glioma Cells Is Sensitive to the State of Microtubule Assembly
Authors: Hough Christopher , Fukamauchi Fumahiko , Chuang De‐Maw
Published: July 2010

Abstract: Microtubule disrupter, colchicine, and microtubule stabilizer, taxol, were used to determine whether microtubules play a role in β‐adrenergic receptor mRNA homeostasis and agonist‐induced down‐regulation in C6 glioma cells. Colchicine treatment had significant, differential, time‐dependent effects on constitutive β1‐ and β2‐adrenergic receptor mRNA levels. These effects stemmed from the action of colchicine on microtubules, because β‐lumicolchicine, an inactive isomer, had no effect, and nocodazole, a structurally unrelated microtubule disrupter, had similar effects. Colchicine treatment had little effect on the total number of β‐adrenergic receptor binding sites as measured by (−)‐[125I]iodopindolol binding, but did alter the relative proportion of β1‐ and β2‐adrenergic receptor subtypes. Colchicine also had no effect on basal cyclic AMP levels. In contrast to colchicine, taxol treatment had little long‐term effect on either β1‐ or β2‐adrenergic receptor mRNA levels. Taxol antagonized the effects of colchicine on total binding and mRNA levels. Taxol treatment increased basal cyclic AMP levels fourfold and potentiated (−)‐isoproterenol‐induced cyclic AMP production. Colchicine pretreatment completely inhibited (−)‐isoproterenol‐induced down‐regulation of β1‐adrenergic receptor mRNA, but not that of β2‐adrenergic receptor mRNA. Taxol pretreatment had little effect on isoproterenol‐induced β‐adrenergic receptor mRNA down‐regulation. Colchicine pretreatment also attenuated isoproterenol‐induced receptor down‐regulation and inhibited agonist‐stimulated cyclic AMP production. These effects of colchicine were antagonized by taxol. Whereas the effects of taxol and colchicine on isoproterenol‐induced down‐regulation of β‐adrenergic receptor mRNA are consistent with their effects on cyclic AMP production, those of colchicine in the absence of stimulation must involve other mechanisms. The data demonstrate that the state of microtubule assembly can affect cyclic AMP levels, β1‐ and β2‐adrenergic receptor mRNA, and binding site levels in C6 glioma cells.
Characterization of an Atypical Member of the Na+/Cl−‐Dependent Transporter Family: Chromosomal Localization and Distribution in GABAergic and Glutamatergic Neurons in the Rat Brain
Authors: El Mestikawy Salah , Giros Bruno , Pohl Michel , Hamon Michel , Kingsmore Stephen F. , Seldin Michael F. , Caron Marc G.
Published: July 2010

Abstract: A 3.7‐kb cDNA fragment, designated rat‐XT1, was isolated from a rat whole‐brain cDNA library. The nucleotide sequence of XT1 codes for a 727 amino acid protein with a calculated molecular mass of 81,139 Da and 12 putative transmembrane domains. This protein shares significant homology (28–32%) with the monoamine‐ (dopamine, norepinephrine, serotonin), amino acid‐ (taurine, proline, GABA, glycine), choline‐, and betaine‐, Na+/Cl−‐dependent transporters. The homology is especially high within the first, second, sixth, and eighth transmembrane domains (45–75%). Thus, XT1 clearly belongs to the Na+/Cl−‐dependent neurotransmitter transporter superfamily. However, XT1 may define a new subfamily of transporter because it differs structurally from other members of this family in that the extracellular loop linking transmembrane domains 7 and 8 and the C‐terminal tail are significantly larger in size. Transient or stable expression of rat‐XT1 failed to confer to the transfected cells the ability to transport actively any of the >60 established or putative neurotransmitter substances assessed. Northern blot analyses of peripheral and neural tissues demonstrated that expression of the 8‐kb XT1 mRNA is essentially restricted to the nervous system. In situ hybridization demonstrated a broad but discrete localization of XT1 message in the CNS, particularly in the cerebellum (Purkinje and granular cell layers), the hippocampus (pyramidal and granular cell layers), and the thalamus and throughout the cerebral cortex. This distribution parallels that of the neurotransmitters glutamate and aspartate; however, neither of these excitatory amino acids is a substrate for transport. One noticeable exception to the codistribution of the mRNA for rat‐XT1 and these excitatory neurotransmitters is the cerebellar Purkinje cell layer, in which GABAergic neurons are localized. The gene encoding for XT1 is localized to the mouse chromosome 3 in the vicinity of the locus for the mouse neurological disorder spastic (spa).
The Addition of Glucose‐1‐Phosphate to the Cytoplasmic Glycoprotein Phosphoglucomutase Is Modulated by Intracellular Calcium in PC12 Cells and Rat Cortical Synaptosomes
Authors: Veyna Nancy A. , Jay Jeanne C. , Srisomsap Chantragan , Bounelis Pam , Marchase Richard B.
Published: July 2010

Abstract: In a number of different cell types, phosphorylation of a 63‐kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus‐sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester‐linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [14C]glucose or the [β‐35S]phosphorothioate analogue of UDP‐glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.