bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptosis Induced by Differentiation or Serum Deprivation in an Immortalized Central Nervous System Neuronal Cell Line
Authors: Eves Eva M. , Boise Lawrence H. , Thompson Craig B. , Wagner Andrew J. , Hay Nissim , Rosner Marsha R.
Published: July 2010

Abstract: To characterize the nature of programmed cell death (PCD) induced in neuronal cells during development, three regulators of apoptosis were investigated: one, the bcl‐2‐related genes, modulate cell survival, and the other two, the interleukin‐1β converting enzyme (ICE)‐related enzymes and the tumor suppressor protein p53, have been implicated as mediators of apoptosis. These regulators were studied in H19‐7 cells, an SV40 Tts‐immortalized rat hippocampal neuronal cell line that can be differentiated with basic fibroblast growth factor at the nonpermissive temperature, resulting in a rapid attrition of cells by apoptosis. PCD occurred by two mechanisms in H19‐7 cells: The first was initiated by removal of serum from undifferentiated cells, and the second was a consequence of neuronal differentiation. In differentiated H19‐7 cells, the survival time was increased by both human bcl‐2 and bcl‐xL, and this could be reversed by bcl‐xS.Addition of a peptide inhibitor of the ICE enzyme family to H19‐7 cells resulted in a transient protection against differentiation‐associated apoptosis, whereas no further protection was observed in the BCL‐2‐ or BCL‐XL‐expressing cells. Shifting the differentiated cells to 33°C to inactivate p53 did not significantly affect the apoptotic process, indicating that apoptosis induced by neuronal differentiation is not dependent on the continued presence of p53. By contrast, in undifferentiated cells, cell loss induced by transfer to serum‐free media occurred more rapidly on inactivation of large T, consistent with p53 involvement. This medium‐induced decrease in cell survival could not be rescued by the ICE inhibitor but was partially rescued by BCL‐2 or BCL‐XL. Furthermore, studies involving expression of BCL‐2 and BCL‐XL alone or together revealed differences in the survival dependent on the cellular environment. These results suggest that apoptosis of neuronal cellsoccurs by at least two processes: one in undifferentiated cells initiated by removal of serum and one linked to differentiation. The data implicate the ICE enzyme family but not p53 in apoptosis induced by differentiation and demonstrate that either BCL‐2 or BCL‐XL can prolong the survival of differentiated neuronal cells.
Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of “Impaired” Cholinergic Presynaptic Function
Authors: Marchi Mario , Raiteri Maurizio
Published: July 2010

Abstract: The existence in the mammalian CNS of release‐inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [3H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [3H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [3H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [3H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [3H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [3H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [3H]ACh release; this inhibition was reversed to an enhancement when the external [Ca2+] was lowered. The same occurred when, at 1.2 mM Ca2+, external [K+] was decreased. Oxotremorine still inhibited [3H]ACh release at 0.1 mM Ca2+. When muscarinic receptors were inactivated with atropine, the K+ (15 mM)‐evoked release of [3H]ACh (at 0.1 mM Ca2+) was potently enhanced by ACh acting at nicotinic receptors (EC50⋍ 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.
Colocalization of Glucagon‐Like Peptide‐1 (GLP‐1) Receptors, Glucose Transporter GLUT‐2, and Glucokinase mRNAs in Rat Hypothalamic Cells: Evidence for a Role of GLP‐1 Receptor Agonists as an Inhibitory Signal for Food and Water Intake
Authors: Navarro Miguel , De Fonseca Fernando Rodriguez , Alvarez Elvira , Chowen Julie A. , Zueco Jose A. , Gomez Raquel , Eng John , Blázquez Enrique
Published: July 2010

Abstract: This study was designed to determine the possible role of brain glucagon‐like peptide‐1 (GLP‐1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP‐1 receptors, glucokinase, and GLUT‐2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose‐sensitive neurons mediating feeding behavior. Because GLP‐1 receptors, GLUT‐2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic β cells, the colocalization of specific GLP‐1 receptors and glucose sensing‐related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP‐1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP‐1 (7–36) amide did not modify food and water intake, although a dose‐dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP‐1 (7–36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP‐1 (1–37), a peptide with lower biological activity than GLP‐1 (7–36) amide, did not change feeding behavior in food‐deprived animals. Exendin‐4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP‐1 (7–36) amide. It decreased, in a dose‐dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP‐1 receptor antagonist [exendin (9–39); 2,500 ng] reversed the inhibitory effects of GLP‐1 (7–36) amide (1,000 ng dose) and exendin‐4 (25 ng dose) on food and water ingestion. These findings suggest that GLP‐1 (7–36) amide may modulate both food and drink intake in the rat through a central mechanism.
Prevention of Hyperoxia‐Induced Alterations in Synaptosomal Membrane‐Associated Proteins by N‐tert‐Butyl‐α‐Phenylnitrone and 4‐Hydroxy‐2,2,6,6‐Tetramethylpiperidin‐1‐oxyl (Tempol)
Authors: Howard Beverly J. , Yatin Servet , Hensley Kenneth , Allen Kerry L. , Kelly Jennie P. , Carney John , Butterfield D. Allan
Published: July 2010

Abstract: Hyperoxia has been considered a model of free radical reactive oxygen species production in aging and age‐related disorders. Previously, we studied the membrane protein alterations that occur during hyperoxia; we found that exposure of young animals to 24 h of hyperoxia provided the greatest degree of oxidation of cortical synaptosomal membrane proteins. We reasoned that free radical oxidation was involved in this protein oxidation. In accordance, in the current study we investigated the protective nature of two known free radical scavengers, N‐tert‐butyl‐α‐phenylnitrone (PBN) and 4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (Tempol), against 24‐h hyperoxia damage. The three techniques used in this study were electron paramagnetic resonance (EPR) protein‐specific spin labeling, assay of the activity of the oxidatively sensitive enzyme glutamine synthetase (GS), and measurement of protein carbonyl content. Before hyperoxia, gerbils received intraperitoneal injections of varying concentrations of either of the two free radical scavengers. After 30 min, the gerbils were exposed to 90–100% O2 for 24 h. For the spin labeling experiments, cortical synaptosomes were isolated from gerbils. The membrane proteins were spin labeled with the thiol‐specific label MAL‐6 (2,2,6,6‐tetramethyl‐4‐maleimidopiperidin‐1‐oxyl). As in our earlier study, the EPR spectral parameter of MAL‐6‐labeled membranes, the W/S ratio, decreased with hyperoxia (p < 0.00001). This effect was lessened significantly with administration of PBN (p < 0.0003) or Tempol (p < 0.00003). For the GS and protein carbonyl assays, cortical proteins were used. The activity of the GS decreased with hyperoxia (p < 0.000005), and this effect likewise was lessened with administration of PBN (p < 0.004) or Tempol (p < 0.002). The protein carbonyl content increased with hyperoxia (p < 0.0002), and there was a protective effect found with Tempol (p < 0.000001). The optimum doses for PBN and Tempol were 20 and 5 mg/kg, respectively. The results are discussed with reference to the use of free radical scavengers as potential antiaging agents.
In Vivo Tryptophan Hydroxylase Activity in Rat Major Cerebral Arteries Is Decreased by Dorsal Raphe Nucleus Lesions
Authors: López de Pablo Angel Luis , Moreno María Jesús , Marco Emilio J.
Published: July 2010

Abstract: The in vivo presence of tryptophan hydroxylase activity in rat major cerebral arteries as well as the possible origin of the structure containing it were explored. Enzyme activity was appraised by accumulation of 5‐hydroxytryptophan after inhibition of aromatic l‐amino acid decarboxylase. Decarboxylase inhibition evoked a significant increase in 5‐hydroxytryptophan levels in rat cerebral arteries, striatum, hippocampus, hypothalamus, and plasma but had no effect on aorta. p‐Chlorophenylalanine reduced 5‐hydroxytryptophan accumulation in the cerebral vessels and brain nuclei, whereas α‐methyltyrosine did not modify it except in hypothalamus, where it was enhanced. α‐Methyltyrosine significantly reduced noradrenaline levels in cerebral arteries and l‐dopa accumulation after inhibition of the decarboxylase in striatum. Dorsal raphe nucleus lesioning significantly diminished 5‐hydroxytryptophan formation in cerebral arteries, striatum, and hypothalamus, without affecting it in hippocampus. Lesion of median raphe nucleus reduced 5‐hydroxytryptophan accumulation in hippocampus and in hypothalamus but not in cerebral blood vessels or striatum. Superior cervical ganglia removal decreased noradrenaline levels in cerebral blood vessels without affecting 5‐hydroxytryptophan accumulation. These results indicate the presence of a functionally active tryptophan hydroxylase in rat cerebral arteries associated with fibers originating from dorsal raphe nucleus. This supports that rat major cerebral arteries receive serotonergic innervation from central origin.
p‐Chlorophenylalanine‐Induced Alteration of Somatodendritic Levels of Tryptophan Hydroxylase Within the Rat Mesencephalic Raphe Nuclei
Authors: Raison Sylvie , Rousset Colette , Pujol Jean‐François , Weissmann Dinah
Published: July 2010

Abstract: Tryptophan hydroxylase distribution was examined across the nuclei raphe dorsalis, medianus, and pontis of the adult rat, under basal conditions and 2 days after a single injection of p‐chlorophenylalanine, an irreversible tryptophan hydroxylase inhibitor. Tryptophan hydroxylase‐expressing cells were numbered in transverse sections processed for immunohistochemistry, and the area of tryptophan hydroxylase distribution was delineated in adjacent sections transferred onto nitrocellulose and processed for immunoautoradiography. Two distinct areas were visualized: an inner zone, corresponding to the area displaying tryptophan hydroxylase‐immunoreactive cells (so‐called somatic area), and an outer zone, here called perisomatic, devoid of perikarya yet rich in tryptophan hydroxylase‐positive neuropil in the histological sections. After treatment with p‐chlorophenylalanine, a significant decrease in the number of tryptophan hydroxylase‐immunoreactive cells could be observed only in the rostral raphe dorsalis, particularly within its ventromedian and dorsomedian subdivisions. In all raphe nuclei, the topological reconstruction of the somatic area was not modified. Based on the densitometric measurements in the immunoautoradiographs, however, a dramatic decrease in the content, concentration, and volume of expression of tryptophan hydroxylase could be documented in the three raphe nuclei. Detailed analysis of these results led to the conclusion that (a) tryptophan hydroxylase expression is differentially regulated in different serotoninergic cell body subpopulations of the raphe, some of which are more sensitive to p‐chlorophenylalanine, and (b) distribution of tryptophan hydroxylase protein is modified also in the somatodendritic area in all raphe nuclei.
Unsaturated Free Fatty Acids Increase Benzodiazepine Receptor Agonist Binding Depending on the Subunit Composition of the GABAA Receptor Complex
Authors: Witt M. R. , Westh‐Hansen S. E. , Rasmussen P. Baad , Hastrup S. , Nielsen M.
Published: July 2010

Abstract: It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (αxβyγ2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus‐transfected Sf9 insect cell system. At 10−4M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (>200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the α3β2γ2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to α1βxγ2 and α2βxγ2 receptor combinations were observed, and weak effects (130% of control values) were detected using the α5β2γ2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an α1β2γ2 receptor combination was used. Given the heterogeneity of the GABAA/benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.
Preparation of Giant Myelin Vesicles and Proteoliposomes to Register Ionic Channels
Authors: Regueiro Pilar , Monreal Jaime , Díaz Ricardo S. , Sierra Felipe
Published: July 2010

Abstract: Myelin vesicles, reconstituted liposomes with proteolipid protein (PLP), the main protein component of myelin, and electrophysiological patch‐clamp are potentially powerful tools to study the role of myelin in functional ionic channels. However, technical difficulties in the vesiculation of myelin and the small size of the vesicles obtained do not permit the application of micropipettes for current recordings. From a suspension of purified myelin we have prepared oligolamellar vesicles (mean diameter of 144 nm) using the so‐called French pressure system. From this preparation we obtained giant myelin vesicles ∼10 µm in mean diameter, using a dehydration‐rehydration procedure. Qualitative analysis of proteins by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed no significant loss of any component in these vesicles due to pressure, in comparison with non‐vesiculated myelin. A way of preparing giant liposomes of ∼80–100 µm and proteoliposomes of ∼30 µm in mean diameter, using the same dehydration‐rehydration procedure, is also reported. Reconstitution of purified PLP in giant liposomes was confirmed by fluorescent labeling of PLP and by fluorescence microscopy. The current recordings from these vesicles prove the validity of these methods and provide significant evidence of the existence of ionic channels in myelin membranes and the possibility that PLP functions as a channel. The physiological significance and characterization of these channels remain yet unresolved. These results have a special significance for elucidating the molecular role of myelin in the regulation of neural activity and in the brain ion microenvironment.
Characterisation and Molecular Identification of Adrenomedullin Binding Sites in the Rat Spinal Cord: A Comparison with Calcitonin Gene‐Related Peptide Receptors
Authors: Owji Ali A. , Gardiner James V. , Upton Paul D. , Mahmoodi Mehdi , Ghatei Mohammad A. , Bloom Stephen R. , Smith David M.
Published: July 2010

Abstract: Calcitonin gene‐related peptide (CGRP) and its receptors are found in mammalian spinal cord. We show, for the first time, binding sites for the novel related peptide adrenomedullin in rat spinal cord microsomes. 125I‐Adrenomedullin binding showed high affinity (KD = 0.45 ± 0.06 nM) and sites were abundant (Bmax = 723 ± 71 fmol/mg of protein). CGRP, amylin, and calcitonin did not compete at these sites (Ki > 10 µM). High‐affinity CGRP binding sites (KD = 0.18 ± 0.01 nM) were much less numerous (Bmax = 17.7 ± 2.4 fmol/mg of protein) and showed competition by unlabeled adrenomedullin (Ki = 34.6 ± 2.4 nM). Chemical cross‐linking revealed a major band for 125I‐adrenomedullin of Mr = 84,400 ± 1,200 and a minor band of Mr = 122,000 ± 8,700. 125I‐CGRP cross‐linking showed bands of lower molecular weight (Mr = 74,500 ± 5,000 and 61,000 ± 2,200). Enzymic deglycosylation of the adrenomedullin binding site showed a considerable carbohydrate content. Neither adrenomedullin nor CGRP was able to increase cyclic AMP in spinal cord. Adrenomedullin mRNA was present in spinal cord, at one‐third of its level in lung, and adrenomedullin immunoreactivity was present, at a low concentration (40 fmol/g of tissue). Thus, the presence of abundant binding sites and adrenomedullin mRNA and immunoreactivity anticipate an as yet undefined function for this peptide in spinal cord.
Cyclic AMP Inhibits Activation of Mitogen‐Activated Protein Kinase and Cell Proliferation in Response to Growth Factors in Cultured Rat Cortical Astrocytes
Authors: Kurino Masahito , Fukunaga Kohji , Ushio Yukitaka , Miyamoto Eishichi
Published: July 2010

Abstract: The cyclic AMP (cAMP)‐induced inhibitory effect on cell proliferation was examined through inhibition of mitogen‐activated protein kinase (MAP kinase) activation in cultured rat cortical astrocytes. Basic fibroblast growth factor (bFGF) at 10 ng/ml maximally stimulated MAP kinase activity, which peaks during 10 min and prolonged for 24 h. Likewise, DNA synthesis was maximally potentiated with 10 ng/ml bFGF and correlated with MAP kinase activity in a dose‐dependent manner. Dibutyryl cAMP (dbcAMP) at 1 mM and isoproterenol at 10 µM inhibited MAP kinase activation and DNA synthesis potentiation with bFGF and platelet‐derived growth factor to the control level in cultured astrocytes and C6 glioma cells. The stimulation with bFGF caused a prominent translocation of MAP kinase from the cytosol to the nucleus after 1 h in astrocytes. Treatment of the cells with dbcAMP and isoproterenol completely prevented the translocation of MAP kinase. In experiments with 32P‐labeled cultured astrocytes, phosphorylation of Raf‐1 was apparently stimulated with bFGF. Treatment with dbcAMP or isoproterenol had a greatly inhibitory effect on the stimulation of Raf‐1 phosphorylation with bFGF. Consistent with the effect on Raf‐1 phosphorylation, dbcAMP and isoproterenol completely prevented bFGF‐induced phosphorylation of MAP kinase kinases, target proteins of Raf‐1. Our observations suggest that cAMP‐induced suppression of cell growth in astrocytes is due to the inhibitory effect on activation of MAP kinase and its translocation to the nucleus and that the site of the cAMP action is located at Raf‐1 or the upstream site of Raf‐1.
Regional and Ontogenic Expression of the NMDA Receptor Subunit NR2D Protein in Rat Brain Using a Subunit‐Specific Antibody
Authors: Dunah Anthone W. , Yasuda Robert P. , Wang Yue‐hua , Luo Jianhong , Dávila‐García Martha I. , Gbadegesin Muyiwa , Vicini Stefano , Wolfe Barry B.
Published: July 2010

Abstract: A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an ∼160‐kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross‐reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N‐glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.
Two Components of Glutamate Exocytosis Differentially Affected by Presynaptic Modulation
Authors: Herrero I. , Castro E. , Miras‐Portugal M. T. , Sánchez‐Prieto J.
Published: July 2010

Abstract: The total Ca2+‐dependent release of glutamate induced by depolarization of cerebrocortical nerve terminals with KCl was analyzed into a fast and a slow component. The fast component exhibited a decay time of <1 s and accounted for 0.95 ± 0.10 nmol of glutamate, whereas the slow component, which exhibited a decay time of 52 ± 7 s, accounted for the release of 2.48 ± 0.19 nmol of glutamate. These two components were differentially affected by the Ca2+ chelator BAPTA, the divalent cation Sr2+, or the botulinum neurotoxin A. The adenosine A1 receptor agonist N6‐cyclohexyladenosine strongly reduced the fast component without altering the slow component. In contrast, the inhibitory effect of arachidonic acid and the facilitatory action of the metabotropic glutamate receptor agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid were observed as a decrease and an increase, respectively, in the two components. It is concluded, first, that the fast and slow components correspond to the release of docked and mobilized vesicles, respectively, and second, that presynaptic modulation more significantly alters the fast component of release.
Protective Effects of Extracellular Acidosis and Blockade of Sodium/Hydrogen Ion Exchange During Recovery from Metabolic Inhibition in Neuronal Tissue Culture
Authors: Vornov James J. , Thomas Ajit G. , Jo David
Published: July 2010

Abstract: Acidosis is a universal response of tissue to ischemia. In the brain, severe acidosis has been linked to worsening of cerebral infarction. However, milder acidosis can have protective effects. As part of our investigations of the therapeutic window in our neuronal tissue culture model of ischemia, we investigated the effects of acidosis during recovery from brief simulated ischemia. Ischemic conditions were simulated in dissociated cortical cultures by metabolic inhibition with potassium cyanide to block oxidative metabolism and 2‐deoxyglucose to block glycolysis. Lowering the extracellular pH (pHe) to 6.2 during metabolic inhibition had no effect on injury, as measured by lactate dehydrogenase release from cultures after 24 h of recovery. Lowering the pHe during the first hour of recovery, in contrast, had profound protective effects. When the duration of metabolic inhibition was lengthened to 30 min, most of the protective effects of the NMDA receptor antagonist MK‐801 were lost. However, the protective effects of acidosis were unchanged. This suggested that the protective effects of extracellular acidosis could be due to more than blockade of NMDA receptors. Intracellular acidosis might be responsible. To test this, recovery of intracellular pH (pHi) was slowed by incubation with blockers of Na+/H+ exchangers at normal pHe. The two compounds tested, dimethylamiloride and harmaline, had protective effects when present during recovery from metabolic inhibition. Measurements of pHi confirmed that the blockers slowed recovery from intracellular acidosis; more rapid pHi recovery was correlated with injury. The protective effects of acidosis could be reversed by brief incubation with the protonophore monensin, which rapidly normalized pHi. These results are the first demonstration of the protective effects of blocking Na+/H+ exchange in a model of cerebral ischemia. The protective effects of acidosis appear to arise either from suppressing pH‐sensitive mechanisms of injury or from blocking sodium entry due to Na+/H+ exchange.
Alzheimer's Disease Neurofibrillary Tangles Contain Mitosis‐Specific Phosphoepitopes
Authors: Kondratick Christine M. , Vandré Dale D.
Published: July 2010

Abstract: Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimer's disease (AD). PHFs are composed of the microtubule‐associated protein (MAP) τ, which is abnormally phosphorylated in AD. Normal fetal τ is also phosphorylated and shares certain phosphoepitopes with PHF‐τ. The abnormal phosphorylation of PHF‐τ is considered to be involved in the formation of PHFs and subsequent degeneration of AD neurons. We have previously shown that other neuronal MAPs, such as MAP1B, contain mitosis‐specific phosphoepitopes. In addition to mitotic cells, these epitopes are also expressed in fetal brain and PC12 cells during differentiation and neurite outgrowth. One hypothesis regarding the etiology of AD involves the reactivation of a fetal‐like state and mitotic conditions in selected neurons. To determine if similar mitosis‐associated phosphoepitopes appeared in AD, sections of hippocampal tissue were stained for immunoreactivity with antibodies recognizing both τ and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF‐τ antibody stained neurofibrillary lesions and colocalized to pyramidal neurons in AD samples. In addition, PHFs isolated from an AD brain reacted with both antibodies. The MPM2 antibody specifically reacted with τ in the isolated PHF fraction but not normal adult τ. In addition, MPM2 failed to react with normal fetal or adult τ obtained from rat brains. The MPM2 antibody also recognized human MAP1B; however, MAP1B was not present in the PHF fraction. Our results indicate that MPM2 recognized a phosphoepitope present on PHF‐τ. Because normal fetal or adult rat brain τ did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.
Glial Fibrillary Acidic Protein Expression in Rat Brain and in Radial Glia Culture Is Delayed by Prenatal Ethanol Exposure
Authors: Vallés S. , Sancho‐Tello M. , Miñana R. , Climent E. , Renau‐Piqueras J. , Guerri C.
Published: July 2010

Abstract: The alterations in astrocyte proliferation and differentiation induced by prenatal exposure to alcohol (PEA) suggest that ethanol exposure affects the radial glial cells, the main astrocytic precursors. We have investigated the effects of ethanol on the early stages of astrogliogenesis by analyzing the developmental pattern of vimentin and glial fibrillary acidic protein (GFAP) immunoreactivity and their mRNA levels during embryonic/fetal brain development and in radial glia in primary culture. GFAP appeared late in gestation and at day 5 of culture of radial glial, whereas GFAP mRNA was first detected on fetal day 15 and increased in content on fetal day 21. In contrast, the levels of vimentin and its mRNA were high at fetal day 15 but decreased on day 21. Alcohol exposure delays the appearance of GFAP and its mRNA and significantly decreases the GFAP expression in fetal brain and in primary culture of radial glial. In addition, some morphological alterations were observed in PEA glial cells in culture. These results demonstrate that astroglial precursor cells are damaged by prenatal exposure to ethanol and suggest that abnormalities in the astrogliogenesis may underlie the disruption in neuronal migration and other CNS alterations observed after prenatal ethanol exposure.
Role of Nitric Oxide in Methamphetamine Neurotoxicity: Protection by 7‐Nitroindazole, an Inhibitor of Neuronal Nitric Oxide Synthase
Authors: Di Monte Donato A. , Royland Joyce E. , Jakowec Michael W. , Langston J. William
Published: July 2010

Abstract: The role of nitric oxide (NO•) in the neurotoxic effects of methamphetamine (METH) was evaluated using 7‐nitroindazole (7‐NI), a potent inhibitor of neuronal nitric oxide synthase. Treatment of mice with 7‐NI (50 mg/kg) almost completely counteracted the loss of dopamine, 3,4‐dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity observed 5 days after four injections of 10 or 7.5 mg/kg METH. With the higher dose of METH, this protection at 5 days occurred despite the fact that combined administration of METH and 7‐NI significantly increased lethality and exacerbated METH‐induced dopamine release (as indicated by a greater dopamine depletion at 90 min and 1 day). Combined treatment with 4 × 10 mg/kg METH and 7‐NI also slightly increased the body temperature of mice as compared with METH alone. Thus, the neuroprotective effects of 7‐NI are independent from lethality, are not likely to be related to a reduction of METH‐induced dopamine release, and are not due to a decrease in body temperature. These results indicate that NO• formation is an important step leading to METH neurotoxicity, and suggest that the cytotoxic properties of NO• may be directly involved in dopaminergic terminal damage.
Interactions of δ‐Conotoxins with Alkaloid Neurotoxins Reveal Differences Between the Silent and Effective Binding Sites on Voltage‐Sensitive Sodium Channels
Authors: Shichor Iris , Fainzilber Michael , Pelhate Marcel , Malecot Clair O. , Zlotkin Eliahu , Gordon Dalia
Published: July 2010

Abstract: The δ‐conotoxin‐TxVIA from Conus textile (δTxVIA) is a mollusk‐specific conotoxin that slows sodium channel inactivation exclusively in mollusk neuronal membranes but reveals high‐affinity binding to both mollusk (effective binding) and rat brain (silent binding) neuronal membranes, despite not having any toxic effect in vertebrates in vivo and in vitro. Using binding studies with radioactive δTxVIA we demonstrate that a different mollusk‐specific conotoxin, δ‐conotoxin‐GmVIA from the venom of Conus gloriamaris, possesses “silent” and effective binding properties in rat brain and mollusk sodium channels, respectively. Binding studies and electrophysiological tests with both vertebrate muscle and insect neuronal preparations have indicated that the silent binding sites of δTxVIA are highly conserved in a wide range of distinct vertebrate and insect sodium channels. Direct probing of receptor site 2 by a tritiated derivative of batrachotoxin ([3H]BTX‐B) revealed that [3H]BTX‐B binding in mollusk sodium channels is of high affinity with no addition of enhancing ligands, unlike [3H]BTX‐B binding in rat brain. In contrast to the negative allosteric modulation of δTxVIA binding by veratridine, δTxVIA is not able to affect the binding of [3H]BTX‐B in mollusk neuronal membranes but reduces [3H]BTX‐B binding in rat brain in the presence of α‐scorpion toxins. The latter finding indicates the existence of a pharmacological distinction between the silent and effective binding sites of δTxVIA and points out possible functionally important structural differences between molluscan and rat brain sodium channels.
Functional Impairment in Protein Kinase C by RACK1 (Receptor for Activated C Kinase 1) Deficiency in Aged Rat Brain Cortex
Authors: Pascale Alessia , Fortino Ida , Govoni Stefano , Trabucchi Marco , Wetsel William C. , Battaini Fiorenzo
Published: July 2010

Abstract: Several laboratories have reported a lack of protein kinase C (PKC) activation in response to various stimuli in the brain of aged rats. It has been suggested that changes in lipid membrane composition could be related to this functional deficit. However, recent evidence has indicated that the translocation of PKC to the different subcellular compartments is controlled by protein‐protein interactions. Recently, a class of proteins, termed receptors for activated C kinase (RACKs), have been described that bind PKC. The present study was conducted to determine whether alterations in RACK1, the best‐characterized member of RACKs, were associated with changes in translocation and expression of PKC. Quantitative immunoblotting revealed that RACK1 content was decreased by ∼50% in aged rat brain cortex, compared with that in adult and middle‐aged animals. The levels of calcium‐independent PKCδ and ε, interacting with RACK1, and related calcium‐independent PKC activity were not modified by the aging process. By comparison, phorbol ester‐stimulated translocation of this activity and of PKCδ and ε immunoreactivity was absent in cortex from aged animals, as well as the translocation of the calcium‐dependent PKCβ, also known to interact with RACK1. These results indicate that a deficit in RACK1 may contribute to the functional impairment in PKC activation observed in aged rat brain.
Characterisation of Human 5‐Hydroxytryptamine2A and 5‐Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH‐SY5Y: Comparative Stimulation by Hallucinogenic Drugs
Authors: Newton R. A. , Phipps S. L. , Flanigan T. P. , Newberry N. R. , Carey J. E. , Kumar C. , McDonald B. , Chen C. , Elliott J. M.
Published: July 2010

Abstract: Stable transfection of the human neuroblastoma cell line SH‐SY5Y with the human 5‐hydroxytryptamine2A (5‐HT2A) or 5‐HT2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5‐HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5‐HT2C receptor (pEC50 = 7.80 ± 0.06) compared with the 5‐HT2A receptor (pEC50 = 7.30 ± 0.08). Activation of the 5‐HT2A receptor caused a transient fourfold increase in intracellular Ca2+ concentration. Whole‐cell recordings of cells clamped at −50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5‐HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d‐lysergic acid diethylamide (LSD), 1‐(4‐iodo‐2,5‐dimethoxyphenyl)‐2‐aminopropane (DOI), 5‐methoxy‐N,N‐dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m‐chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5‐HT revealed selective activation of the 5‐HT2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5‐methoxy‐N,N‐dimethyltryptamine were relatively nonselective, whereas m‐chlorophenylpiperazine selectively activated the 5‐HT2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5‐HT2C receptor, whereas activity at the 5‐HT2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.
Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP‐Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum
Authors: Rich Roger C. , Aswad Dana W.
Published: July 2010

Abstract: We have identified previously a synaptic membrane‐associated protein, PP59, that serves as a substrate for cyclic AMP‐dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X‐100 plus 1 M KCl. A 290‐fold purification of PP59 was achieved by selective solubilization, followed by continuous‐elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP‐dependent protein kinase phosphorylation site within PP59, the partially purified 32P‐phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed‐phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala‐Arg‐Glu‐Arg‐Ser‐Asp‐Ser(P)‐Thr‐Gly‐Ser‐Ser‐Ser‐Val‐Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14‐amino acid sequence was used to develop polyclonal anti‐peptide antibodies that were affinity‐purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP‐dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.
Insulin Promotes the Hydrolysis of a Glycosyl Phosphatidylinositol in Cultured Rat Astroglial Cells
Authors: Ruiz‐Albusac Juan Miguel , Velázquez Esther , Iglesias Javier , Jimenez Encarnacion , Blázquez Enrique
Published: July 2010

Abstract: Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol‐specific phospholipase C and glycosyl phosphatidylinositol‐specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 nM insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1–2 min. In addition, an inverse concentration‐dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C‐mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin‐like growth factor (IGF)‐I and/or IGF‐II receptors, as demonstrated by affinity cross‐linking studies. Also, the effects of 5 nM IGF‐I or 5 nM IGF‐II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet‐derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacyl‐glycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF‐I, and especially with IGF‐II, for 10 min. Despite the remarkable homology among insulin, IGF‐I, and IGF‐II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.
Retinoic Acid Is a Negative Regulator of the Neuropeptide Y/Peptide YY Y1 Receptor Gene in SK‐N‐MC Cells
Authors: Mannon Peter J. , Kaiser Lynn M.
Published: July 2010

Abstract: To identify effectors of neuropeptide Y Y1 receptor gene expression predicted by putative regulatory elements in its 5′ flanking region, we examined the effects of several transcriptionally active agents on Y1 receptor mRNA levels and 125I‐peptide YY binding capacity in SK‐N‐MC cells. Phorbol ester caused a rapid, transient 2.6‐fold increase in Y1R mRNA levels. However, all trans‐retinoic acid caused a rapid, sustained decrease in Y1 receptor mRNA levels (to 25% of control). Cycloheximide pretreatment did not attenuate the maximal inhibitory effect of retinoic acid, but it prolonged the time to achieve maximal efficacy. The retinoic acid effect was secondary to both a significant decrease in Y1 receptor mRNA stability and a decreased Y1 receptor gene transcription rate. Y1 receptor binding capacity was also significantly decreased after retinoic acid treatment (368 ± 25 vs. 496 ± 28 fmol/mg of protein for control). These data support a role for retinoic acid as an important agent regulating Y1 receptor gene expression and mediating Y1 receptor down‐regulation.
Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus
Authors: Charlton Maura E. , Sweetnam Paul M. , Fitzgerald Lawrence W. , Terwilliger Rosemarie Z. , Nestler Eric J. , Duman Ronald S.
Published: July 2010

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABAA receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABAA receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α1 and α5 subunits was regulated by ethanol in a region‐specific and time‐dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α1 and α5 subunit immunoreactivity was reduced. In the VTA, levels of α1 subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α1 subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α5 mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABAA receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.
Calcium/Calmodulin‐Dependent Kinase II Phosphorylates Drosophila Visual Arrestin
Authors: Kahn Esther S. , Matsumoto Hiroyuki
Published: July 2010

Abstract: Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein‐coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor‐specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light‐adaptive response. Type II Ca2+/calmodulin‐dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+/calmodulin‐dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca2+/calmodulin‐dependent kinase II such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+/calmodulin‐dependent kinase II autoinhibitory domain. We also show that Ca2+/calmodulin‐dependent kinase II activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca2+/calmodulin‐dependent kinase II phosphorylates phosrestin I. We suggest that Ca2+/calmodulin‐dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation.
Serotonin Stimulation of 5‐HT4 Receptors Indirectly Enhances In Vivo Dopamine Release in the Rat Striatum
Authors: Deurwaerdère Philippe De , L'hirondel Marie , Bonhomme Norbert , Lucas Guillaume , Cheramy André , Spampinato Umberto
Published: July 2010

Abstract: Serotonin (5‐HT) applied at 1, 3, and 10 µM into the striatum of halothane‐anesthetized rats by in vivo microdialysis enhanced dopamine (DA) outflow up to 173, 283, and 584% of baseline values, respectively. The 5‐HT effect was partially reduced by 1 or 10 µM GR 125,487, a 5‐HT4 antagonist, and by 100 µM DAU 6285, a 5‐HT3/4 antagonist, whereas the 5‐HT1/2/6 antagonist methiothepin (50 µM) was ineffective. In the presence of tetrodotoxin the effect of 1 µM 5‐HT was not affected by 5‐HT4 antagonists. In addition, tetrodotoxin abolished the increase in DA release induced by the 5‐HT4 agonist (S)‐zacopride (100 µM). In striatal synaptosomes, 1 and 10 µM 5‐HT increased the outflow of newly synthesized [3H]DA up to 163 and 635% of control values, respectively. The 5‐HT4 agonists BIMU 8 and (S)‐zacopride (1 and 10 µM) failed to modify [3H]DA outflow, whereas 5‐methoxytryptamine (5‐MeOT) at 10 µM increased it (62%). In prelabeled [3H]DA synaptosomes, 1 µM 5‐HT, but not (S)‐zacopride (1 and 10 µM), increased [3H]DA outflow. DAU 6285 (10 µM) failed to modify the enhancement of newly synthesized [3H]DA outflow induced by 5‐MeOT or 5‐HT (1 µM), whereas the effect of 5‐HT was reduced to the same extent by the DA reuptake inhibitor nomifensine (1 µM) alone or in the presence of DAU 6285. These results show that striatal 5‐HT4 receptors are involved in the 5‐HT‐induced enhancement of striatal DA release in vivo and that they are not located on striatal DA terminals.