bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for the Action of Endogenous Adenosine in the Rabbit Retina: Modulation of the Light‐Evoked Release of Acetylcholine
Authors: Blazynski Christine , Woods Cynthia , Mathews Gregory C.
Published: October 2006

Abstract: Much evidence has accumulated supporting the hypothesis that the purine nucleoside adenosine may indeed function as a neuromodulator in the mammalian retina, but to date no reports have directly illustrated a physiological role for this nucleoside. In other regions of the CNS, adenosine agonists decrease transmitter release, whereas antagonists increase release. A similar role for adenosine in the retina is now apparent. The cholinergic amacrine cells of the rabbit retina were labeled with [3H]choline, and the effects of enzymatic adenosine degradation or adenosine antagonists on the light‐evoked efflux of acetylcholine were evaluated. When endogenous adenosine was degraded by addition of adenosine deaminase, the light‐evoked release of radioactivity derived from [3H]choline was significantly increased compared with control values. A similar response was observed when rabbit eyecups were superfused with a selective adenosine A1 receptor antagonist. The effect elicited by adenosine deaminase could be almost completely reversed by addition of cyclo‐pentyladenosine, a highly selective A1 receptor agonist. These effects were observed in either the presence or the absence of picrotoxin. The results demonstrate a modulation of retinal physiology by adenosine.
Acute Reserpine Treatment Increases Rat Brain Serotonin Synthesis Via a Nerve Impulse‐Dependent Mechanism
Authors: Hjorth Stephan
Published: October 2006

Abstract: Notwithstanding recent advances in the understanding of central serotonin (5‐HT) function, important basic aspects of the control of brain 5‐HT neuronal transmission remain incompletely elucidated. The present experiments addressed the putative mechanism(s) by which acute reserpine treatment stimulates cerebral 5‐HT synthesis; also, such studies might shed further light on the relation between impulse flow and transmitter synthesis in central 5‐HT neurons. Reserpine (5 mg/kg, i.p., 90 min before death) markedly elevated 5‐HT synthesis [5‐hydroxytryptophan (5‐HTP) accumulation after decarboxylase inhibition by means of NSD 1015] in the limbic, striatal, and cortical rat brain parts. Hemitransection of ascending neuronal connections between the brainstem and the forebrain, performed immediately before reserpine injection, did not affect the 5‐HT synthesis per se but completely prevented the drug response on the lesioned side. Similarly, systemic administration of the selective 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (0.1 mg/kg, s.c., 60 min before death) blocked the reserpine‐induced elevation of brain 5‐HT synthesis. These findings indicate that the reserpine‐induced rise in axon terminal 5‐HTP accumulation is dependent on intact (5‐HT) neuronal impulse flow, which may or may not involve a transient impairment of somatodendritic 5‐HT release and, in turn, autoreceptor tone.
Synapsin I Regulates Glutamate Release from Rat Brain Synaptosomes
Authors: Nichols Robert A. , Chilcote Tamie J. , Czernik Andrew J. , Greengard Paul
Published: October 2006

Abstract: Introduction of the dephosphorylated form of synapsin 1 into rat brain synaptosomes using freeze‐thaw (transient) perme‐abilization significantly decreased the K+‐induced release of glutamate. In contrast, introduction of synapsin I that had been phosphorylated by Ca2+/calmodulin‐dependent protein kinase II was without effect on glutamate release. Addition of dephosphosynapsin I after freeze‐thaw treatment also had no effect. Thus, the action of synapsin I was dependent on the phosphorylation state of synapsin I and on its entry into the synaptosomes. Our results implicate synapsin I as an important component in the regulation of neurotransmitter release in the mammalian nervous system.
Depression of Mesolimbic Dopamine Transmission and Sensitization to Morphine During Opiate Abstinence
Authors: Acquas Elio , Di Chiara Gaetano
Published: October 2006

Abstract: To investigate the role of mesolimbic dopamine (DA) in the mechanism of drug dependence, extracellular DA was monitored by transcerebral dialysis in the caudal nucleus accumbens under basal conditions and after challenge with morphine (5 mg/kg s.c.) in control rats and in rats made dependent on and then deprived of morphine. Withdrawal from morphine resulted in a marked reduction of extracellular DA concentrations from control values at 1, 2, 3, and 5 days of withdrawal. After 7 days of withdrawal, DA output was less, but still significantly, reduced. Challenge with morphine resulted in stimulation of DA output in controls (maximum, 35%), no effect on the first day of withdrawal, and stimulation similar to controls’ on days 2 and 7 of withdrawal. On day 5 and, particularly, on day 3 of withdrawal, morphine‐induced stimulation of DA output was markedly potentiated (maximum, 100 and 160%, respectively). Changes in the sensitivity of DA transmission to morphine challenge were associated with changes in the behavioral stimulant effects of morphine, with tolerance on day 1 and marked sensitization on days 3 and 5 but also on day 7, when morphine‐induced stimulation of transmission was no longer potentiated. The results indicate that repeated morphine administration induces a state of dependence in DA neurons and a short‐lasting tolerance followed by an increased sensitivity to its stimulant effects on DA transmission. These changes might play an important role in the development of opiate addiction and in the maintenance of opiate self‐administration in dependent subjects.
Postinjury Changes in Platelet‐Derived Growth Factor‐Like Activity in Fish and Rat Optic Nerves
Authors: Lotan Mirit , Schwartz Michal
Published: October 2006

Abstract: The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet‐derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF‐responsive cells. The results suggest that PDGF‐like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF‐like levels. The results are discussed with respect to the possible role of PDGF in regeneration.
Long‐Term Activation of Protein Kinase C by Nicotine in Bovine Adrenal Chromaffin Cells
Authors: Tuominen R. K. , McMillian M. K. , Ye H. , Stachowiak M. K. , Hudson P. M. , Hong J. S.
Published: October 2006

Abstract: Previous results from our laboratory suggest that long‐term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12‐myristate 13‐acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine‐synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long‐term effects. Nicotine increased particulate PKC activity in a concentration‐dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1‐dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine‐receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13‐dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic‐receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine‐stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic‐receptor activation increases PKC activity and immunoreactivity in BAM cells. The long‐term PKC activation may serve several functions, such as activation of mRNA production and a negative feedback regulation of either nicotinic receptors or voltage‐dependent Ca2+ channels.
Age‐Dependent Changes in the Oligosaccharide Structure of the Major Myelin Glycoprotein, P0
Authors: Brunden Kurt R.
Published: October 2006

Abstract: The single oligosaccharide moiety of the major myelin glycoprotein, P0, resides in an immunoglobulin‐like domain that appears to participate in homophilic binding. The studies presented here indicate that the structure of the P0 oligosaccharide from rat nerve changes as a function of Schwann cell age. Examination of 5‐day‐old nerve revealed that P0 contained predominantly endo‐β‐β‐acetylglucosa‐minidase H (endo H)‐resistant, complex‐type oligosaccharide. In contrast, P0 from adult rats had mostly endo H‐sensitive carbohydrate, indicating the presence of appreciable high‐mannose and/or hybrid‐type oligosaccharide on the glycoprotein. The endo H‐sensitive and resistant P0 of adult nerve could be readily phosphorylated by protein kinase C, as could the complex‐type P0 from 5‐day‐old nerve. This suggests that the glycoprotein progresses to the plasma membrane and myelin regardless of the type of oligosaccharide chain. Analysis of 35SO42‐labeled P0 showed that the sulfate group was found on both endo H‐sensitive and ‐resistant oligosaccharide. The endo H‐sensitive P0 carbohydrate from adult nerve appears to be primarily of the hybrid type, as evidenced by (a) the elution profile of [3H]mannose‐labeled P0 glycopeptides from adult nerve during concanavalin A chromatography and (b) the inability of P0 from adult nerve to interact with Galanthus nivalis agglutinin. The observed age‐dependent changes of P0 oligosaccharide may modify the binding properties of this myelin glycoprotein.
Purification and Partial Structural and Functional Characterization of Mouse Myelin/Oligodendrocyte Glycoprotein
Authors: Amiguet Patrick , Gardinier Minnetta V. , Zanetta Jean‐Pierre , Matthieu Jean‐Marie
Published: October 2006

Abstract: The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8‐18C5 identifies MOG. Several studies have shown that anti‐MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25‐kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase, indicating that there are no or few O‐linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N‐terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal‐transducing GTP‐binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.
Calcium Independence of Phosphoinositide Hydrolysis‐Induced Increase in Cyclic AMP Accumulation in SK‐N‐SH Human Neuroblastoma Cells
Authors: Baumgold Jesse , Paek Robert , Fiskum Gary
Published: October 2006

Abstract: Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin‐sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)‐mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK‐N‐SH human neuroblastoma cells while blocking the IP3‐mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) abolished the carbachol‐mediated intracellular calcium release without abolishing the carbachol‐mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist‐induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor‐mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.
Simultaneous Measurement of Extracellular Morphine and Serotonin in Brain Tissue and CSF by Microdialysis in Awake Rats
Authors: Matos F. Fátima , Rollema Hans , Basbaum Allan I.
Published: October 2006

Abstract: In this report, we describe an HPLC with electrochemical detection assay for the simultaneous measurement of levels of morphine, serotonin, 5‐hydroxyindole‐3‐acetic acid, and homovanillic acid in dialysates of various brain areas and CSF in the awake rat. Morphine could be detected in the dialysates after a single intraperitoneal injection, with doses as low as 1.0 mg/kg. The time course of extracellular morphine content in the lateral hypothalamus, striatum, cerebellum, periaqueductal gray, and dorsal horn of the spinal cord and in CSF, from the ventricles and cisterna magna, was similar. We detected morphine in the first 15‐min sample, and levels peaked 45–60 min after injection. Maximal dialysate levels, however, varied with the type of dialysis probe used and the area sampled. The most efficient in vivo recovery was in CSF dialysates from the cisterna magna, presumably because of minimal tissue interference with the dialysis probe. For this reason, the cisterna is an ideal region for sampling CSF. Morphine had no significant effect on the extracellular concentrations of serotonin in any of the areas studied and did not modify or only slightly increased levels of tissue metabolites; however, morphine markedly increased the CSF levels of 5‐hydroxyindole‐3‐acetic acid and homovanillic acid. Because microdialysis in freely moving animals permits assessment of the behavioral effects of morphine while continuously monitoring the drug levels in discrete brain regions, this approach will greatly facilitate future studies of the neurochemical basis of morphine's effects in the brain.
Regional and Temporal Alterations in Ca2+/Calmodulin‐Dependent Protein Kinase II and Calcineurin in the Hippocampus of Rat Brain After Transient Forebrain Ischemia
Authors: Morioka Motohiro , Fukunaga Kohji , Yasugawa Setsuko , Nagahiro Shinji , Ushio Yukitaka , Miyamoto Eishichi
Published: October 2006

Abstract: We have investigated regional and temporal alterations in Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin‐dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6–12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin‐independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin‐dependent protein phosphorylation‐dephosphorylation may occur.
N‐Methyl‐D‐Aspartate Antagonists Block Fos‐Like Protein Expression Induced via Multiple Signaling Pathways in Cultured Cortical Neurons
Authors: Hisanaga Kinya , Sagar Stephen M. , Sharp Frank R.
Published: October 2006

Abstract: c‐fos mRNA and Fos‐like protein(s) (FLP) are induced in cultured cortical neurons by glutamate, high K+, phorbol ester, basic fibroblast growth factor, Zn2+, and vasoactive intestinal peptide. Glutamate induction of c‐fos mRNA and FLP is blocked by noncompetitive N‐methyl‐D‐aspartate (NMDA) antagonist, MK‐801, and competitive NMDA antagonists, 4‐(3‐phosphonopropyl)piperazin‐2‐carboxylic acid and 2‐amino‐7‐phosphonoheptanoate. These antagonists partially block high K+‐, phorbol ester‐, Zn2+‐, and VIP‐induced c‐fos mRNA expression, but have no effect on bFGF‐induced c‐fos mRNA expression. However, both competitive and noncompetitive NMDA antagonists completely block FLP induction by all of these agents without affecting total protein synthesis. Therefore, these NMDA antagonists block FLP translation, without blocking c‐fos transcription. It is hypothesized that NMDA receptor activation is required for translation of c‐fos mRNA in cortical neurons after stimulation of multiple intracellular signaling pathways. It is possible that NMDA antagonists prevent cortical plasticity by blocking induction of the Fos protein that would normally be induced by neurotrophic factors, neurotransmitters, and neuromodulators.
Increased Expression of β‐Amyloid Protein Precursor and Microtubule‐Associated Protein τ During the Differentiation of Murine Embryonal Carcinoma Cells
Authors: Fukuchi Ken‐ichiro , Deeb Samir S. , Kamino Kouzin , Ogburn Charles E. , Snow Alan D. , Sekiguchi Raymond T. , Wight Thomas N. , Piussan Henri , Martin George M.
Published: October 2006

Abstract: Expression of the genes encoding the β/A4 amyloid protein precursor (APP) and microtubule‐associated protein τ was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34‐ (mRNA) and 50‐fold (protein) during neuronal differentiation; APP‐695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of τ mRNA. Moreover, subsequent decreases in the levels of APP and τ mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that >85% of the P19‐derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and τ protein in development of Alzheimer's disease in cholinergic neurons.
Estrogen Induction of Cytochrome c Oxidase Subunit III in Rat Hippocampus
Authors: Bettini Ezio , Maggi Adriana
Published: October 2006

Abstract: Differential screening of a cDNA library prepared from mRNA of the hippocampus of estrogen‐stimulated ovariectomized female rats led to the identification of a single estrogen‐induced clone. Analysis of the sequence identified this cDNA as the gene coding for subunit III of the enzyme cytochrome c oxidase. Cytochrome c oxidase subunit III mRNA levels significantly increased as early as 3 h following the administration of a single dose of hormone. This effect was visible in the hippocampus and in the hypothalamus, but not in the other brain areas examined. Because subunit III of the cytochrome c oxidase is of mitochondrial origin, the mechanism involved in the estrogenic effect is still unknown. The observation that the activity of cytochrome c oxidase can also be induced by estrogens in the hippocampus indicates that this induction may be secondary to the increased expression of the other subunits of cytochrome c oxidase or to the general increase of neuronal activity.
The DM20 Protein of Myelin: Intracellular and Surface Expression Patterns in Transfectants
Authors: Timsit S. , Sinoway M. P. , Levy L. , Allinquant B. , Stempak J. , Staugaitis S. M. , Colman D. R.
Published: October 2006

Abstract: DM20 is an abundant CNS myelin‐specific protein whose role in myelinogenesis is unknown. We have cloned the DM20 cDNA from adult mouse brain total RNA using the polymerase chain reaction and expressed it in HeLa cells. DM20, detected by immunofluorescence in stable transfectants, is present in some cells in large, intensely fluorescent intracellular clumps that probably represent elements of the rough endoplasmic reticulum and Golgi apparatus. Frequently, intense DM20 fluorescence could be detected at the plasma membrane. These findings are consistent with previous studies demonstrating that an intracellular “pool” of DM20 and its larger isoform, proteolipid protein, exists and that a substantial lag occurs between synthesis and insertion of these proteins into the expanding myelin membrane. Permanent DM20 expressors in contact with one another do not display any ultrastructural rearrangements at regions of cell–cell contact, in contrast to what we have previously reported for P0, a PNS‐specific protein shown to mediate adhesion of the extracellular faces of the Schwann cell during PNS myelinogenesis. We believe that these results indicate that if DM20 is indeed an adhesion molecule, this property is likely to be significantly more subtle than P0‐mediated adhesion.
Detergent‐Insoluble Cortical Lewy Body Fibrils Share Epitopes with Neurofilament and τ
Authors: Pollanen Michael S. , Bergeron Catherine , Weyer Luitgard
Published: October 2006

Abstract: Lewy bodies are cytoskeletal inclusions associated with neuronal injury and death in idiopathic Parkinson's disease and other neurodegenerative disorders. The chemical composition of the 8–10‐nm fibrils of the Lewy body is unknown, although they are related to both normal cytoskeletal elements and paired helical filaments of Alzheimer neurofibrillary tangles. From the Lewy body‐rich cerebral cortex of patients with diffuse Lewy body disease we have isolated intact Lewy bodies using a high salt buffer/nonionic detergent gradient centrifugation procedure and extracted the constitutive fibrils with urea and sodium dodecyl sulfate. Urea/detergent‐resistant Lewy body fibrils were solubilized with formic acid and found to contain a single protein band of 68 kDa, which was not found in identically prepared normal brain homogenates. The Lewy body derived‐polypeptide was recognized on immunoblots by a polyclonal antibody that reacted with both the 68‐kDa neurofilament subunit and the microtubule‐associated protein τ. The 68‐kDa Lewy body protein was not labeled by the monoclonal antibody τ‐1 despite prior in vitro enzymatic dephosphorylation. We conclude that the detergent‐insoluble component of the cortical Lewy body fibril shares epitopes with neurofilament and τ and may be a posttranslationally modified derivative of either neurofilament or τ with substantially altered biochemical and immunologic properties.
Inhibition of Forskolin‐Stimulated Cyclic AMP Formation by 1‐Aminocyclopentane‐trans‐1,3‐Dicarboxylate in Guinea‐Pig Cerebral Cortical Slices
Authors: Cartmell J. , Kemp J. A. , Alexander S. P. H. , Hill S. J. , Kendall D. A.
Published: October 2006

Abstract: The effects of the selective metabotropic glutamate receptor agonist 1‐aminocyclopentane‐trans‐1,3‐dicarboxylate (t‐ACPD) on forskolin‐stimulated cyclic AMP formation in guinea‐pig cerebral cortex slices were determined. t‐ACPD inhibited the accumulation of [3H]cyclic AMP by ∼80%, with an IC50 value of 35 ± 4 μM. The effect was reversible and stereoselective, with the 1S,3R isomer being ∼400‐fold more potent than the 1R,3S isomer. L‐Glutamate (over a restricted concentration range) also partially inhibited the response to forskolin, but quisqualate, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA), and N‐methyl‐D‐aspartate (NMDA) were ineffective. The effect of t‐ACPD was not blocked by antagonists of the phospholipase C‐linked metabotropic glutamate receptor, the AMPA ionotropic glutamate receptor, or the NMDA receptor. In summary, our results indicate the presence of a glutamate receptor in guinea‐pig brain that is activated selectively by t‐ACPD and that is negatively linked to adenylyl cyclase.
1‐Methyl‐4‐Phenylpyridinium Produces Excitotoxic Lesions in Rat Striatum as a Result of Impairment of Oxidative Metabolism
Authors: Storey Elsdon , Hyman Bradley T. , Jenkins Bruce , Brouillet Emmanuel , Miller Joanne M. , Rosen Bruce R. , Beal M. Flint
Published: October 2006

Abstract: The effects of 1‐methyl‐4‐phenylpyridinium (MPP+) were studied in rat striatum. Using freeze‐clamp, microwave, and water‐suppressed proton chemical shift magnetic resonance imaging techniques, MPP+ resulted in marked increases in lactate and a depletion of ATP for up to 48 h after the injections. MPP+ produced dose‐dependent depletions of dopamine, serotonin, γ‐aminobutyric acid, and substance P that were partially blocked at 1 week by prior decortication or completely blocked by MK‐801 at 24 h. The lesions showed relative sparing of somatostatin‐neuropeptide Y neurons, consistent with N‐methyl‐D‐aspartate (NMDA) excitotoxicity. MPP+ produces impairment of oxidative phosphorylation in vivo, which may result in membrane depolarization with persistent activation of NMDA receptors and excitotoxic neuronal degeneration. An impairment of energy metabolism may therefore underlie slow excitotoxic neuronal death in neurodegenerative diseases.
Effects of Choline and Quiescence on Drosophila Choline Acetyltransferase Expression and Acetylcholine Production by Transduced Rat Fibroblasts
Authors: Schinstine M. , Rosenberg M. B. , Routledge‐Ward C. , Friedmann T. , Gage F. H.
Published: October 2006

Abstract: Rat‐1 fibroblasts were transduced to express Drosophila choline acetyltransferase. The presence of an active enzyme in these cells (Rat‐1/dChAT) was confirmed using various methods. Rat‐1/dChAT fibroblasts released acetylcholine (ACh) into the culture medium. Moreover, intra‐and extracellular levels of ACh could be increased by adding exogenous choline chloride. In addition, serum starvation or confluence‐induced quiescence caused an 80% decrease in recombinant choline acetyltransferase activity (compared with actively growing cells). ACh release was also repressed in quiescent fibroblast cultures. Exogenous choline could mitigate the decrease in ACh release. These results indicate that Rat‐1 fibroblasts can be genetically modified to produce ACh and that ACh release can be controlled by introducing choline into the culture medium. Furthermore, these data demonstrate that the expression of the retroviral promoter used in this study decreases with the onset of quiescence; however, exogenous choline can increase the amount of ACh released by quiescent fibroblasts.
Characterisation of an Allosteric Modulatory Protein Associated with α‐[3H]Amino‐3‐Hydroxy‐5‐Methylisoxazolepropionate Binding Sites in Chick Telencephalon: Effects of High‐Energy Radiation and Detergent Solubilisation
Authors: Henley Jeremy M. , Nielsen Mogens , Barnard Eric A.
Published: October 2006

Abstract: α‐[3H]Amino‐3‐hydroxy‐5‐methylisoxazolepro‐pionate ([3H]AMPA) binds to 1‐day‐old chick telencephalon membranes with KD and Bmax values of 138 nM and 2.56 pmol/mg of protein, respectively. High‐energy radiation bombardment of intact frozen telencephalon resulted in a biphasic inactivation curve for [3H]AMPA binding. At a 5.8‐Mrad radiation dose, the affinity of [3H]AMPA binding was increased (54 nM), but there was no apparent alteration in the Bmax value (2.76 pmol/mg of protein). We attribute this phenomenon to the inactivation of a high molecular weight modulatory protein that down‐regulates the affinity of [3H]AMPA binding. The estimated molecular masses of the AMPA binding site and of the modulatory component were 59 and 108 kDa, respectively. Solubilisation with n‐octyl‐β‐glucopyranoside resulted in an increase in the Bmax (4.7 pmol/ mg of protein) with no pronounced alteration in the affinity (109 nM) of [3H]AMPA binding. However, the solubilisation‐induced increase in Bmax did not occur in telencephalon irradiated before solubilisation. In contrast, the increase in affinity induced by radiation treatment was still detected in solubilised extracts. These results suggest that the number and affinity of [3H]AMPA sites in chick telencephalon are closely regulated and that the modulatory systems involved are affected by both irradiation and solubilisation.
High‐Affinity Choline Transport Regulation by Drug Administration During Postnatal Development
Authors: Happe H. K. , Murrin L. C.
Published: October 2006

Abstract: High‐affinity choline transport sites specifically bind [3H]hemicholinium‐3. Hemicholinium‐3 binding sites are regulated by in vivo drug treatments in the same manner as these drugs alter acetylcholine release and high‐affinity choline transport. The current study examines regulation of binding sites by in vivo drug administration for adult, day 15, and day 5 rats. Drugs or saline were administered intraperito‐neally, and striatal and cortical membrane preparations were assayed. Control [3H]hemicholinium‐3 binding increases twofold between postnatal days 5 and 15 only in striatum. After day 15, binding increases 2.7‐fold in cortex and striatum. Nicotine treatment increases striatal and cortical hemicholinium‐3 binding at all three ages, with greater percent increases at day 5. Haloperidol increases binding only in striatum, again with larger effects at day 5. Both striatal and cortical binding are reduced by oxotremorine; however, the magnitude of this effect is unchanged during development. Pentobarbital reduces binding only in striatum, with no developmental change. Atropine and apomorphine do not change binding from control values. In summary, all drug treatments effective in adults were already effective by day 5. Cholinergic terminals present early in development are regulated by similar nicotinic and muscarinic cholinergic, dopaminergic, and sedative‐hypnotic mechanisms as the adult. Changes in magnitude may be due to changes in drug metabolism or to developmental differences in regulation.
Synthesis of Glial Fibrillary Acidic Protein in Rat C6 Glioma in Chemically Defined Medium: Cyclic AMP‐Dependent Transcriptional and Translational Regulation
Authors: Messens Joris , Slegers Herman
Published: October 2006

Abstract: Glial fibrillary acidic protein (GFA) expression was induced in rat C6 glioma in chemically defined medium by the addition of N6,O2′‐dibutyryl cyclic AMP (dbcAMP). Induction was dependent on the increase in intracellular cyclic AMP (cAMP), which was linearly correlated with added dbcAMP. Contrary to GFA mRNA synthesis, which can be obtained by cAMP‐dependent and ‐independent pathways, translation of mRNA into GFA was observed only above a cellular cAMP concentration of approximately 0.2 fmol/cell. dbcAMP stimulation did not affect the vimentin concentration, which remained at a low level, but changed the cellular morphology from a bipolar to a stellate shape. A similar morphological change was observed after stimulation of C6 with lipopolysaccharide (LPS). However, LPS did not significantly increase the intracellular concentration of cAMP and the LPS‐induced mRNA was not translated into GFA. Our results indicate that GFA synthesis is regulated at the mRNA level and at the translational level and that a cAMP‐dependent mechanism determines the ultimate synthesis of GFA by a yet unknown mechanism.
Regulation of Tyrosine Hydroxylase and Dopamine β‐Hydroxylase mRNA Levels in Rat Adrenals by a Single and Repeated Immobilization Stress
Authors: McMahon Anne , Kvetňansk Richard , Fukuhara Koki , Weise Virginia K. , Kopin Irwin J. , Sabban Esther L.
Published: October 2006

Abstract: Adrenal catecholamines are known to mediate many of the physiological consequences of the “fight or flight” response to stress. However, the mechanisms by which the long‐term responses to repeated stress are mediated are less well understood and possibly involve alterations in gene expression. In this study the effects of a single and repeated immobilization stress on mRNA levels of the adrenal catecholamine biosynthetic enzymes, tyrosine hydroxylase and dopamine β‐hydroxylase, were examined. A repeated 2‐hr daily immobilization for 7 consecutive days markedly elevated both tyrosine hydroxylase and dopamine β‐hydroxylase mRNA levels (about six‐ and fourfold, respectively). In contrast, tyrosine hydroxylase but not dopamine β‐hydroxylase mRNA levels were elevated immediately following a single immobilization. The elevation in tyrosine hydroxylase mRNA with a single immobilization was as high as with seven daily repeated immobilizations. This elevation was not sustained and returned toward control values 24 hr later. Both tyrosine hydroxylase and dopamine β‐hydroxylase mRNA levels were elevated immediately following two daily immobilizations to levels similar to those observed after seven immobilizations and were maintained 24 hr later. The results indicate that both tyrosine hydroxylase and dopamine β‐hydroxylase mRNA levels are elevated by stress; however, the mechanism and/or timing of their regulation are not identical.
A Dihydropyridine‐Resistant Component in the Rat Adrenal Secretory Response to Splanchnic Nerve Stimulation
Authors: López M. G. , Shukla R. , García A. G. , Wakade A. R.
Published: October 2006

Abstract: A study of the effects of dihydropyridine Ca2+ channel modulators on the release of catecholamines from perfused rat adrenal glands, evoked by electrical stimulation of their splanchnic nerves, is presented. Electrically mediated secretory responses were compared to chemically mediated responses (exogenous acetylcholine, nicotine, or high K+). Intensities of stimuli were selected to produce quantitatively similar secretory responses (between 100 and 200 ng per stimulus). The main finding of the study is that responses to transmural stimulation (300 pulses at 1 or 10 Hz) and to acetylcholine were inhibited only partially (about 50%) by isradipine, an L‐type Ca2+ channel blocker. In contrast, responses to high K+ (17.5 mM for 2 min) were highly sensitive to isradipine (IC50= 8.2 nM). Responses to nicotine were also fully inhibited by this drug. Bay K 8644 (an L‐type Ca2+ channel activator) potentiated mildly the secretory responses to electrical stimulation at 10 Hz and to acetylcholine, but increased threefold the responses to K+ and nicotine. It is, therefore, likely that responses mediated by high K+ or nicotinic receptors are triggered by external Ca2+ gaining access to the internal secretory machinery through L‐type, dihydropyridine‐sensitive voltage‐dependent Ca2+ channels. However, in addition to nicotinic receptors, the physiological stimulation of adrenal medulla chromaffin cells through splanchnic nerves has other components, i.e., muscarinic receptor stimulation or the release of cotransmitters such as vasoactive intestinal polypeptide. The poorer sensitivity to dihydropyridines of secretory responses triggered by electrical stimulation of splanchnic nerve terminals or exogenous acetylcholine speaks in favor of alternative Ca2+ pathways, probably some dihydropyri‐dine‐resistant Ca2+ channels, in modulating the physiological adrenal catecholamine secretory process.
Concerted CMP‐Dependent [3H]Inositol Labeling of Phosphoinositides and Agonist Activation of Phospholipase C in Rat Brain Cortical Membranes
Authors: Claro Enrique , Wallace Michael A. , Fain John N.
Published: October 2006

Abstract: [3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP‐dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl‐inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP‐dependent or independent fashions were hydrolyzed by guan‐osine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐ and carbachol‐stimulated phospholipase C. Unlike CMP‐dependent labeling, however, CMP‐independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxy‐cholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the Ptdlns synthase‐catalyzed reaction because of the presence of deoxy‐cholate required to observe carbachol‐stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo‐[3H]inositol 1‐mono‐ phosphate was minimal and myo‐[3H]inositol 1,4‐bisphos‐phate was the predominant product. Although PtdIns labeling and 3Hpolyphosphoinositide formation were unaffected by GTPγS and carbachol and had no or little lag period, GTPγS‐ and carbachol‐stimulated appearance of 3H‐Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H‐Ins phosphates was restricted to a narrow range of free calcium concentrations (10–300 nM). These results show the concerted activities of Ptdlns synthase, Ptdlns 4‐kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide‐dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.
Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve
Authors: Wong Elsie , Mizisin Andrew P. , Garrett Robert S. , Miller Arnold L. , Powell Henry C.
Published: October 2006

Abstract: The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two‐ and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.
Proligodendroblast Antigen (POA), a Developmental Antigen Expressed by A007/O4‐Positive Oligodendrocyte Progenitors Prior to the Appearance of Sulfatide and Galactocerebroside
Authors: Bansal Rashmi , Stefansson K. , Pfeiffer S. E.
Published: October 2006

Abstract: Evidence is presented for the immunological identification of a developmental antigen appearing at a critical point in the oligodendroglial lineage. Specifically, monoclonal antibody A007 recognizes cells in the oligodendrocyte lineage at two distinct stages. Analyses of purified lipid standards and lipid extracts from galactocerebroside‐positive (GalC+) oligodendrocytes by enzyme‐linked immunosorbent assay, lipid dot blot, and immuno‐TLC demonstrated that A007 recognizes sulfatide (SUL) and seminolipid. However, neither 35SO4 incorporation into SUL nor SUL accumulation could be detected in A007‐positive cells lacking galactocerebroside (i.e., A007+GalC− progenitor cells) present early in development. These data suggest that A007 also recognizes an antigen, named proligodendroblast antigen (POA), that appears during the late stage of oligodendrocyte progenitor development prior to the expression by oligodendrocytes of SUL and GalC. We have previously reported that monoclonal antibody O4 also recognizes not only SUL and seminolipid, but in addition an antigen that appears prior to the expression of SUL and galactocerebroside. In the present study all A007+ cells were also O4+ (and vice versa), and the developmental patterns of the two antibodies appeared to be identical. We conclude that (1) A007 is similar or identical to O4 with respect to its antigenic specificity, and (2) during oligodendrocyte lineage progression both antibodies react first with antigen POA on the surface of the oligodendrocyte progenitor cell prior to the expression of SUL [i.e., A007+O4+(POA+)SUL−GalC− proligodendroblasts], and only later with SUL as terminally differentiating oligodendrocytes emerge (i.e., A007+‐O4+SUL+GalC+ oligodendrocytes).
Modulation of Extracellular γ‐Aminobutyric Acid in the Ventral Pallidum Using In Vivo Microdialysis
Authors: Bourdelais Andrea J. , Kalivas Peter W.
Published: October 2006

Abstract: Intracranial microdialysis was used to investigate the origin of extracellular γ‐aminobutyric acid (GABA) in the ventral pallidum. Changes in basal GABA levels in response to membrane depolarizers, ion‐channel blockers, and receptor agonists were determined. Antagonism of Ca2+ fluxes with high Mg2+ in a Ca2+‐free perfusion buffer decreased GABA levels by up to 30%. Inhibition of voltage‐dependent Na+ channels by the addition of tetrodotoxin also significantly decreased basal extracellular GABA concentrations by up to 45%, and blockade of Ca2+ and Na+ channels with verapamil reduced extracellular GABA by as much as 30%. The addition of either the GABAA agonist, muscimol, or the GABAB agonist, baclofen, produced a 40% reduction in extracellular GABA. GABA release was stimulated by high K+ and the addition of veratridine to increase Na+ influx. High K+‐induced release was predominately Ca2+‐dependent, whereas the effect of veratridine was potentiated in the absence of extracellular Ca2+. Both high K+‐ and veratridine‐induced elevations in extracellular GABA were inhibited by baclofen, whereas only veratridine‐induced release was antagonized by muscimol. These results demonstrate that at least 50% of basal extracellular GABA in the ventral pallidum is derived from Ca2+‐ or Na+‐dependent mechanisms. They also suggest that Na+‐dependent release of GABA via reversal of the uptake carrier can be shown in vivo.
The NK‐1 Receptor and a Calcium‐Phospholipid Pathway: Inositol Trisphosphate Production and Calcium Movements Induced by Selective Agonists of Neurokinin Receptors in Rat Parotid Glands
Authors: Guillemain Isabelle , Rollandy Isabelle , Imhoff Viviane , Rossignol Bernard
Published: October 2006

Abstract: In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium‐phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK‐1 receptor agonist, [Sar9Met(O2)11]‐substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK‐2 and NK‐3 receptor agonists, [β‐Ala8]‐NKA(4‐10) and [MePhe7]‐NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK‐1 receptors are coupled to the calcium‐phospholipid pathway. The C‐terminal part of substance P appeared to be sufficient to stimulate this route because the C‐terminal octapeptide, substance P(4‐l 1), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK‐2 and NK‐3 receptors, if present in the rat parotid glands, are not associated with the calcium‐phospholipid pathway.
Regional Effects of Sodium Valproate on Extracellular Concentrations of 5‐Hydroxytryptamine, Dopamine, and Their Metabolites in the Rat Brain: An In Vivo Microdialysis Study
Authors: Biggs C. S. , Pearce B. R. , Fowler L. J. , Whitton P. S.
Published: October 2006

Abstract: The effects of sodium valproate (VPA; 100, 200, and 400 mg/kg, i.p.) on ventral hippocampal and anterior caudate putamen extracellular levels ofdopamine (DA) and 5‐hydroxytryptamine (5‐HT) were examined using in vivo microdialysis. VPA induced dose‐related increases in dialysate DA, 3,4‐dihydroxyphenylacetic acid, and 5‐HT in the ventral hippocampus. Anterior caudate putamen dialysate 5‐HT was also dose dependently elevated by the drug, whereas DA levels tended to decrease with increasing VPA dose. In contrast, VPA (200, 400, and 800 mg/kg, i.p.) produced no significant elevation of DA in posterior caudate putamen dialysates, although 5‐HT levels were significantly elevated at the 400‐ and 800‐mg/kg doses. In all three regions studied, dialysate concentrations of 5‐hydroxyindoleacetic acid and homovanillic acid remained at basal levels following VPA treatments. The results are discussed with regard to the possible anticonvulsant mode of action of VPA.
Reversibility of Nimodipine Binding to Brain in Transient Cerebral Ischemia
Authors: Hogan Matthew J. , Hakim Antoine M.
Published: October 2006

Abstract: Using autoradiography, we have measured the in vivo binding of [3H]nimodipine to brain in a rat model of reversible cerebral ischemia. Ischemia was induced by simultaneous occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery by microaneurysm clips. Rats were studied after 15 min of ischemia (ischemic group) or after 45 min of reperfusion following 15 min of ischemia (reperfused group). Regional cerebral blood flow (CBF) was determined autoradiographically using [14C]iodoantipyrine in both ischemic (n = 6) and reperfused (n = 6) groups. During ischemia blood flow in the territory of the MCA was depressed and recovered to normal only in the distal territory of the MCA following reperfusion. [3H]Nimodipine binding in the ischemic group (n = 12) was elevated in ischemic brain regions and declined significantly (p < 0.01) in these regions in the reperfused group (n = 11). The ratio of the volume of cortex showing increased binding to the total volume of the forebrain was 0.113 pmn 0.025 (mean pmn SD) in the ischemic group and declined to 0.080 pmn 0.027 following reperfusion (p < 0.005). In general, infarct was only observed in regions showing persistent elevation of nimodipine binding following reperfusion as determined by histology performed in a separate group of rats (n = 8) after 24 h of reperfusion. We conclude that increased nimodipine binding to ischemic tissue is initially reversible with prompt reestablishment of CBF and is a sensitive indicator of early and reversible ischemia‐induced cerebral dysfunction.