bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamatergic Activation of Hippocampal Phospholipase D: Postnatal Fading and Receptor Desensitization
Authors: Klein Jochen , Vakil Marjan , Bergman Frits , Holler Thomas , Iovino Mario , Löffelholz Konrad
Published: July 2010

Abstract: Phospholipase D (PLD) activity was determined in rat hippocampal slices between postnatal days 3 and 35. After birth, basal PLD activity was low and, within 2 weeks, increased to reach a plateau that was maintained up to the adult age. Likewise the response to glutamate developed postnatally to reach a maximum at day 8, but then faded rapidly and was almost absent at day 35. Activation of PLD by 4β‐phorbol 12β,13α‐dibutyrate (PDB) was independent of age, whereas the effect of aluminum fluoride (AlF4−) increased to a plateau within the first week. At day 8, PLD stimulation by glutamate via metabotropic receptors involved protein kinase C activation, but was independent of Ca2+ influx; the time course of PLD activation by PDB or AlF4− was linear throughout the experiment, whereas the response to glutamate or 1‐aminocyclopentane‐1,3‐dicarboxylic acid followed a biphasic pattern: the rapid “first phase activation” desensitized within a few minutes and disclosed a small, but maintained “second phase.” Pretreatment experiments confirmed desensitization of PLD activation by glutamate, but not by AlF4− or PDB. The biphasic pattern of glutamatergic PLD activation changed during development, i.e., the first phase activation faded and the second phase remained. These results were fully confirmed by the time courses of the PLD‐mediated efflux of choline evoked by glutamate. In conclusion, postnatal glutamatergic activation of hippocampal PLD is composed of a pronounced and desensitizing first phase activation and a small, but nondesensitizing second phase. The first, but not the second, phase activation fades rapidly during development. The hypothesis is discussed that the glutamatergic activation of PLD occurs along different pathways in neonate and adult tissue.
Effects of Copper on Survival of Prion Protein Knockout Neurons and Glia
Authors: Brown David R. , Schmidt Bernhard , Kretzschmar Hans A.
Published: July 2010

Abstract: The N‐terminal region of the prion protein (PrP) contains an octameric repeat region suggested to bind copper. A 32‐amino acid peptide (PrPOcta) based on this region in the protein was tested for its effects on cultured cerebellar cells. Cerebellar cells from mice deficient in cellular PrP (Prnp0/0 mice) are more sensitive to copper toxicity and oxidative stress. PrPOcta selectively promotes the survival of Prnp0/0 cerebellar cells. However, PrPOcta also reduces the toxicity of CuSO4 on cerebellar cells and abolishes the difference in increased sensitivity of Prnp0/0 cells to both copper toxicity and also oxidative stress from xanthine oxidase. PrPOcta does not promote the survival or proliferation of astrocytes or microglia. The survival‐promoting effects of PrPOcta on neurons may be due to its ability to effectively chelate copper. The octameric repeat region of PrP may represent a functional domain of the native protein.
Histamine H1 Receptor Antagonists Produce Increases in Extracellular Acetylcholine in Rat Frontal Cortex and Hippocampus
Authors: Dringenberg Hans C. , De Souza‐Silva M. Angelica , Roßmüller Jens , Huston Joseph P. , Schwarting Rainer K. W.
Published: July 2010

Abstract: Lesions of the neuronal histaminergic system or pharmacological blockade of histamine receptors, e.g., with histamine H1 receptor antagonists, can enhance the performance of rats in several tests of learning and memory. The underlying neuronal systems that mediate these behavioral effects are not known. Here, we examined the effects of treatment with histamine H1 antagonists on extracellular levels of acetylcholine (ACh) in adult rats anesthetized with urethane (1.25 g/kg). ACh was quantified using in vivo microdialysis and HPLC with electrochemical detection. Basal levels of ACh in the frontal cortex and hippocampus were in the range of 0.54 ± 0.13 and 0.96 ± 0.17 pmol/20 min, respectively. Injection (intraperitoneally) of saline did not produce significant increases in ACh levels, even though there was a slight and gradual increase in cortical ACh levels throughout the course of the experiments (up to 4 h after an injection). Administration of the H1 receptor antagonist chlorpheniramine (intraperitoneally) produced a dose‐dependent increase of cortical ACh levels to a maximum of 260, 280, and 570% of baseline values after doses of 5, 10, and 20 mg/kg, respectively. In the hippocampus, ACh content increased to a maximum of ∼600% of baseline levels after chlorpheniramine administration (20 mg/kg, i.p.). Administration of the H1 antagonist pyrilamine (intraperitoneally) increased cortical ACh content to a maximum of 300 and 500%, whereas hippocampal ACh levels increased to 215 and 280% after doses of 10 and 20 mg/kg, respectively. In an additional experiment using nonanesthetized, freely moving rats, cortical ACh content showed a moderate increase (to 190%) after saline injections (intraperitoneally) and a much higher increase (to 370%) after chlorpheniramine treatment (20 mg/kg, i.p.). These data suggest that cortical and hippocampal levels of ACh can be effectively modulated by systemic treatment with histamine H1 antagonists. The increases in ACh levels produced by H1 antagonists may suggest that some histaminergic receptors exert an inhibitory influence over central ACh levels. The enhanced availability of ACh in the forebrain may contribute to the behavioral effects observed with H1 antagonist treatment.
Effects of Human Type 1α Metabotropic Glutamate Receptor Expression Level on Phosphoinositide and Ca2+ Signalling in an Inducible Cell Expression System
Authors: Hermans E. , Young K. W. , Challiss R. A. J. , Nahorski S. R.
Published: July 2010

Abstract: Stable expression of the human type 1α metabotropic glutamate (mGlu1α) receptor was achieved in Chinese hamster ovary cells using an isopropyl‐β‐d‐thiogalactoside (IPTG)‐repressible expression system. Treatment of the cells with IPTG resulted in a time‐ and concentration‐dependent induction of receptor expression. Maximal expression was obtained after treatment of the cells with 100 µM IPTG for 20 h, leading to a marked increase in receptor immunoreactivity detected by western blot, >30‐fold stimulation of 3H‐labelled inositol phosphate (3H‐InsP) production, and a robust increase in intracellular calcium concentration in single cells after stimulation with 20 µM quisqualate. The basal level of 3H‐InsP accumulation in cells induced with IPTG was increased by two‐ to threefold as compared with control cells; however, this basal activity was found to be dependent on glutamate released by the cells into the incubation medium. Following IPTG treatment, stable expression of the mGlu1α receptor was maintained for at least 1 week. Taken together, these results clearly indicate the advantages of working with an inducible expression system when studying the biochemical and pharmacological properties of the human mGlu1α receptor in transfected cells.
The Use of DNase I Hypersensitivity Site Mapping to Identify Regulatory Regions of the Human Cholinergic Gene Locus
Authors: Tanaka Hiromitsu , Zhao Yanming , Wu Donghai , Hersh Louis B.
Published: July 2010

Abstract: The DNase I hypersensitive sites of the human cholinergic gene locus, where both the choline acetyltransferase gene and the vesicular acetylcholine transporter gene are located, were analyzed to detect potential regulatory elements involved in cholinergic‐specific transcriptional activation. From this analysis, seven cholinergic cell line‐specific DNase I hypersensitive sites were found. The majority of these sites correspond to active promoter regions of the gene, including the promoter region of the vesicular acetylcholine transporter gene and the R, N, and M exons of the choline acetyltransferase gene. Two DNase I hypersensitive sites were identified in two noncholinergic cell lines, HeLa and MCF‐7. One corresponds to a previously observed non‐cell‐specific enhancer, whereas the other corresponds to a previously described neuronal restrictive silencer element, NRSE. The NRSE has been shown to repress expression of various neuron‐specific genes, not restricted to cholinergic‐specific genes, in nonneuronal cells. This element can account for repression of the cholinergic gene locus in nonneuronal cells but does not account for repression in noncholinergic neurons. In addition, the chromatin boundary domain of the cholinergic gene locus, which possibly corresponds to the end of the transcription unit, was identified.
A DEVD‐Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation
Authors: D'Mello Santosh R. , Aglieco Fabio , Roberts Melanie R. , Borodezt Kristin , Haycock John W.
Published: July 2010

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCI) to medium containing lower levels of K+ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad‐spectrum caspase inhibitors, tert‐butoxycarbonyl‐Asp·(O‐methyl)·fluoromethyl ketone and benzyloxycarbonyl‐Val‐Ala‐Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N‐acetyl Tyr‐Val‐Ala‐Asp·chloromethyl ketone (YVAD·cmk), an interleukin‐1β converting enzyme‐preferring inhibitor, and N‐acetyl Asp‐Glu‐Val‐Asp·fluoromethyl ketone (DEVD·fmk), a CPP32‐preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin‐1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP‐ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD‐sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+‐deprived granule neurons.
The Nitric Oxide‐Cyclic GMP Pathway Plays an Essential Role in Both Promoting Cell Survival of Cerebellar Granule Cells in Culture and Protecting the Cells Against Ethanol Neurotoxicity
Authors: Pantazis Nicholas J. , West James R. , Dai De
Published: July 2010

Abstract: NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10‐day‐old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol‐induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO‐cyclic GMP pathway is essential for NMDA‐mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with NG‐nitro‐l‐arginine methyl ester eliminated both the NMDA‐mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2′‐(hydroxynitrosohydrazono)bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8‐bromo‐cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol‐induced cell death by a mechanism(s) that involves the NO‐cyclic GMP pathway.
Palmitoylation of Neurofascin at a Site in the Membrane‐Spanning Domain Highly Conserved Among the L1 Family of Cell Adhesion Molecules
Authors: Ren Qun , Bennett Vann
Published: July 2010

Abstract: This report presents the first evidence that a member of the L1 family of nervous system cell‐adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane‐spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine‐1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin‐binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell‐adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low‐density membrane population enriched in signaling molecules. These results indicate a palmitate‐dependent targeting of neurofascin to a specialized membrane microdomain.
Inorganic Pi Increases Neuronal Survival in the Acute Early Phase Following Excitotoxic/Oxidative Insults
Authors: Glinn Michele , Ni Binhui , Irwin Robert P. , Kelley S. W. , Lin Sui‐Zhen , Paul Steven M.
Published: July 2010

Abstract: Inorganic phosphate (Pi) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high‐energy phosphate concentrations, and modulation of cytosolic free [Ca2+]. Cultured fetal rat cortical neurons constitutively import Pi, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular Pi is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular Pi dose‐dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. Pi similarly increased neuronal survival after exposure to kainic acid or H2O2. Pi‐exposed neurons had higher basal intracellular [Pi], [ATP], and [GSH], and slightly lower cytosolic free [Ca2+], compared with Pi‐deprived neurons. Pi‐exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H2O2, compared with Pi‐deprived neurons. These findings demonstrate that changes in extracellular and intracellular Pi can affect neuronal survival after excitotoxic or oxidative insults.
The Transcription Factor NF‐κB Mediates Increases in Calcium Currents and Decreases in NMDA‐ and AMPA/Kainate‐Induced Currents Induced by Tumor Necrosis Factor‐α in Hippocampal Neurons
Authors: Furukawa Katsutoshi , Mattson Mark P.
Published: July 2010

Abstract: Tumor necrosis factor‐α (TNFα) is a cytokine rapidly produced in the brain in response to vigorous neuronal activity and tissue injury. TNFα may protect neurons against excitotoxic and oxidative insults by a mechanism involving activation of the transcription factor NF‐κB. Whole‐cell perforated patch clamp recordings in cultured rat hippocampal neurons showed that long‐term treatment (24–48 h) with TNFα increases Ca2+ current density; pharmacological analysis indicated a major increase in current through L‐type voltage‐dependent calcium channels. Long‐term treatment with TNFα caused a decrease in currents induced by glutamate, NMDA, AMPA, and kainate. Shorter exposures to TNFα (acute; 2 h) did not alter Ca2+ current or glutamate receptor agonist‐induced currents. Ceramide, an intracellular messenger that activates the transcription factor NF‐κB, mimicked the actions of TNFs on Ca2+ current density and currents induced by glutamate receptor agonists. Cotreatment with κB decoy DNA abolished the effects of TNFα on Ca2+ current and excitatory amino acid‐induced currents, demonstrating a requirement for NF‐κB activation in the actions of TNFα. Neurons pretreated with TNFα exhibited increased intracellular Ca2+ concentrations following membrane depolarization but reduced intracellular Ca2+ concentration responses to excitatory amino acids, compared with neurons in untreated control cultures or cultures cotreated with κB decoy DNA. These findings suggest important roles for the transcription factor NF‐κB in modulation of voltage‐dependent calcium channels and glutamate receptors and the many physiological and pathophysiological processes in which these ion channels are involved. Such signaling mechanisms may be particularly important in injury settings such as ischemia or trauma, where TNFα expression is increased and NF‐κB is activated.
Biosynthesis and Metabolism of Native and Oxidized Neuropeptide Y in the Hippocampal Mossy Fiber System
Authors: McCarthy J. Brian , Walker Mary , Pierce Joseph , Camp Patricia , White Jeffrey D.
Published: July 2010

Abstract: Neuropeptide Y (NPY) gene expression is known to be modulated in the mossy fiber projection of hippocampal granule cells following seizure. We investigated NPY biosynthesis and metabolism in an attempt to characterize NPY biochemically as a neurotransmitter in the granule cell mossy fiber projection. NPY biosynthesis was compared in normal control animals and in animals that had experienced a single pentylenetetrazole‐induced seizure. In situ hybridization analysis established the postseizure time course of preproNPY mRNA expression in the hippocampal formation, localizing the majority of increased preproNPY mRNA content to the hilus of the dentate gyrus. Radioimmunoassay analysis of the CA3/mossy fiber terminal subfield confirmed a subsequent increase in NPY peptide content. Biosynthesis of NPY peptide by granule cells and transport to the CA3/mossy fiber subfield was demonstrated by in vivo radiolabel infusion to the dentate gyrus/hilus followed by sequential HPLC purification of identified radiolabeled peptide from the CA3/mossy fiber terminal subfield. Additional in vivo radiolabeling studies revealed a postseizure increase in an unidentified NPY‐like immunoreactive (NPY‐LI) species. HPLC/radioimmunoassay analyses of CA3 subfield tissue extracts comparing normal control animals and pentylenetetrazole‐treated animals confirmed the increased total NPY‐LI, and demonstrated that the increased NPY‐LI was comprised of a minor increase in native NPY and a major increase in the unknown NPY‐LI. Data from subsequent and separate analyses incorporating immunoprecipitation with anti‐C‐terminal flanking peptide of NPY, further HPLC purification, and matrix‐assisted laser desorption/ionization mass spectrometry support the conclusion that the unknown NPY‐LI is methionine sulfoxide NPY. NPY and NPY‐sulfoxide displayed differential calcium sensitivity for release from mossy fiber synaptosomes. Similar to NPY, NPY sulfoxide displayed high‐affinity binding to each of the cloned Y1, Y2, Y4, and Y5 receptor subtypes. Postrelease inactivation of NPY was demonstrated in a mossy fiber synaptosomal preparation. Thus, the present study in combination with previously reported electrophysiological activity of NPY in the CA3 subfield demonstrates that NPY fulfills the classical criteria for a neurotransmitter in the hippocampal granule cell mossy fiber projection, and reveals the presence of two molecular forms of NPY that display differential mechanisms of release while maintaining similar receptor potencies.
Neuroprotective Effects of NMDA Receptor Glycine Recognition Site Antagonism: Dependence on Glycine Concentration
Authors: Pearlstein Robert D. , Beirne Joshua P. , Massey Gary W. , Warner David S.
Published: July 2010

Abstract: High‐affinity NMDA receptor glycine recognition site antagonists protect brain tissue from ischemic damage. The neuroprotective effect of 5‐nitro‐6,7‐dichloro‐2,3‐quinoxalinedione (ACEA 1021), a selective NMDA receptor antagonist with nanomolar affinity for the glycine binding site, was examined in rat cortical mixed neuronal/glial cultures. ACEA 1021 alone did not alter spontaneous lactate dehydrogenase (LDH) release. Treatment with ACEA 1021 (0.1–10 µM) before 500 µM glutamate, 30 µM NMDA, or 300 µM kainate exposure was found to reduce LDH release in a concentration‐dependent fashion. These effects were altered by adding glycine to the medium. Glycine (1 mM) partially reversed the effect of ACEA 1021 on kainate cytotoxicity. Glycine (100 µM–1 mM) completely blocked the effects of ACEA 1021 on glutamate and NMDA cytotoxicity. The glycine concentration that produced a half‐maximal potentiation of excitotoxin‐induced LDH release in the presence of 1.0 µM ACEA 1021 was similar for glutamate and NMDA (18 ± 3 and 29 ± 9 µM, respectively). ACEA 1021 also reduced kainate toxicity in cultures treated with MK‐801. The effects of glycine and ACEA 1021 on glutamate‐induced LDH release were consistent with a model of simple competitive interaction for the strychnine‐insensitive NMDA receptor glycine recognition site, although nonspecific effects at the kainate receptor may be of lesser importance.
Desensitization of the δ‐Opioid Receptor Correlates with Its Phosphorylation in SK‐N‐BE Cells: Involvement of a G Protein‐Coupled Receptor Kinase
Authors: Hasbi Ahmed , Polastron Jocelyne , Allouche Stéphane , Stanasila Laura , Massotte Dominique , Jauzac Philippe
Published: July 2010

Abstract: Phosphorylation of G protein‐coupled receptors is considered an important step during their desensitization. In SK‐N‐BE cells, recently presented as a pertinent model for the studies of the human δ‐opioid receptor, pretreatment with the opioid agonist etorphine increased time‐dependently the rate of phosphorylation of a 51‐kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino‐terminal region of the cloned human δ‐opioid receptor, revealed that it corresponded to the δ‐opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ‐opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ‐opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn2+, known to act as G protein‐coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ‐opioid receptor, that a close link exists between receptor phosphorylation and agonist‐promoted desensitization and that desensitization involves a GRK.
Activation of Microtubule‐Associated Protein Kinase (Erk) and p70 S6 Kinase by D2 Dopamine Receptors
Authors: Welsh Gavin I. , Hall David A. , Warnes Andrew , Strange Philip G. , Proud Christopher G.
Published: July 2010

Abstract: The ability of human and rat D2(short) and D2(long) dopamine receptors to activate microtubule‐associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose‐dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D2 dopamine receptor. Activation of Erk and of p70 S6 kinase via the D2 dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the Gi or Go family. Inhibitors of phosphatidylinositol 3‐kinase (PI 3‐kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3‐kinase‐dependent and ‐independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3‐kinase.
Dorsal Root Ganglia‐Specific Expression of the β‐Galactoside α1,2‐Fucosyltransferase Genes in Rabbits
Authors: Hitoshi Seiji , Kusunoki Susumu , Kanazawa Ichiro , Tsuji Shuichi
Published: July 2010

Abstract: The neurons of dorsal root ganglia (DRG) mediate several sensation modalities. The carbohydrate antigens on DRG neurons differ with the sensation modalities that subsets of neurons convey. Despite the important roles of gangliosides and glycoproteins in neuronal differentiation and neuritogenesis of the mammalian nervous system, little is known about the mechanisms underlying the regulation of glycosylation. We previously demonstrated the expression of H‐blood type antigens (Fucα1, 2Galβ) on rabbit DRG neurons of small diameter and dramatic changes in H antigens during the perinatal period. To investigate the possible biological roles and regulatory mechanisms of H antigens, we recently cloned three types of rabbit α1,2‐fucosyltransferase gene that catalyze the biosynthesis of H antigens. Here, we analyze the expression of these genes, RFT‐I, II, and III, in rabbit DRG. The H‐type α1,2‐fucosyltransferase gene, RFT‐I, was expressed in DRG in late embryos to adult rabbits, as detected on northern blotting. The other two secretor‐type α1,2‐fucosyltransferase genes, RFT‐II and III, were observed to be expressed in late embryonic DRG on RTPCR analysis but were not detectable on northern blotting. The expression of the H‐type α1,2‐fucosyltransferase gene was analyzed by in situ hybridization and was found to be abundant in small‐diameter DRG neurons. These results indicate that the H‐type α1,2‐fucosyltransferase gene plays a major role in the regulation of the H antigen expression in DRG during the perinatal period.
Amyloid β‐Peptide Inhibits High‐Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices
Authors: Kar S. , Issa A. M. , Seto D. , Auld D. S. , Collier B. , Quirion R.
Published: July 2010

Abstract: The characteristic pathological features of the postmortem brain of Alzheimer's disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β‐peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ‐related peptides, under acute conditions, potently inhibit K+‐evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ‐mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high‐affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ1–40 (10−8M) potently inhibited veratridine‐evoked endogenous ACh release from rat hippocampal slices and also decreased the K+‐evoked release potentiated by the nitric oxide‐generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10−14 to 10−6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10−6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.
Structural Requirements for Ligand Interactions at the Benzodiazepine Recognition Site of the GABAA Receptor
Authors: Davies Martin , Bateson Alan N. , Dunn Susan M. J.
Published: July 2010

Abstract: His101 of the GABAA receptor α1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [3H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant α subunits were coexpressed with the rat β2 and γ2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [3H]Ro 15‐4513 bound to all mutant receptors with equal or greater affinity than to the wild‐type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15‐1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15‐1788 and Ro 15‐4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect.
Cloning and Chromosomal Mapping of the Murine Norepinephrine Transporter
Authors: Fritz Jeffery D. , Jayanthi Lankupalle D. , Thoreson Molly A. , Blakely Randy D.
Published: July 2010

Abstract: The norepinephrine (NE) transporter (NET), a target of many clinically prescribed antidepressants, regulates noradrenergic neurotransmission by efficiently clearing NE from synaptic spaces after release. To advance our understanding of NET gene structure, regulation, and potential associations with complex behavioral trait loci, we amplified a mouse norepinephrine transporter (mNET) cDNA from placenta total RNA and utilized mNET probes to isolate and characterize the mNET gene. Inferred translation of the major open reading frame of the mNET cDNA predicts a 617‐amino acid protein with 12 putative membrane‐spanning regions and 94% identity to human NET. The coding exons of the mNET cDNA were found to be spread across >36 kb of 129/Svj genomic DNA, with exon‐intron boundaries bearing consensus gt/ag splice sites. Sequence upstream (202 bp) of the inferred translation initiation site matched the sequence of 5′ rapid amplification of cDNA ends products from brain mRNA with no evidence for intervening introns and is preceded by a TATA box and canonical transcriptional regulatory elements that may play a role in mNET expression in vivo. Probes derived from mNET cDNA identified species‐specific MspI restriction fragment length variations within the mNET gene that were utilized to position the gene (Slc6a5) to murine chromosome 8, one recombinant distal to D8Mit15. This site is within a recently defined quantitative trait locus defined for ethanol sensitivity in LSXSS recombinant inbred mice, Lore4. The status of Slc6a5 as a candidate gene for alcohol sensitivity is discussed with respect to studies noting ethanol‐induced alterations in brain NE receptors, NE receptor‐linked adenylate cyclase, and NE transport.
Cloning, Expression, and Pharmacology of Four Human 5‐Hydroxytryptamine4 Receptor Isoforms Produced by Alternative Splicing in the Carboxyl Terminus
Authors: Blondel Olivier , Gastineau Monique , Dahmoune Yamina , Langlois Michel , Fischmeister Rodolphe
Published: July 2010

Abstract: We report here the molecular cloning of three new splice variants of the human serotonin 5‐hydroxytryptamine4 (h5‐HT4) receptor, which we named h5‐HT4(b), h5‐HT4(c), and h5‐HT4(d). The sequence following the splicing site at Leu358 in the C‐terminal tail of h5‐HT4(b) displays a 74% protein identity with the same region in the long form of the rat 5‐HT4 receptor (r5‐HT4L) but is shorter by 18 amino acids compared to its rat counterpart. The splice variants h5‐HT4(c) and h5‐HT4(d) are the first of their kind to be described in any animal species. The C terminus of h5‐HT4(c) displays a high number of putative phosphorylation sites. The h5‐HT4(d) isoform corresponds to an ultrashort form of the receptor, with a truncation two amino acids after the splicing site. Tissue distribution studies revealed some degree of specificity in the pattern of expression of the different isoforms within the human body. The four splice variants transiently expressed in COS‐7 cells displayed an identical 5‐HT4 pharmacological profile and showed a similar ability to stimulate adenylyl cyclase activity in the presence of 5‐HT. The stimulatory pattern of cyclic AMP formation in response to the 5‐HT4 agonist renzapride was found to be significantly different between h5‐HT4(a) and the other h5‐HT4 isoforms, indicating that the splice variants may differ in the way they trigger the signal transduction cascade following receptor activation.
Evidence of Type I and Type II Transforming Growth Factor‐β Receptors in Central Nervous Tissues: Changes Induced by Focal Cerebral Ischemia
Authors: Vivien Denis , Bernaudin Myriam , Buisson Alain , Divoux Didier , MacKenzie Eric T. , Nouvelot André
Published: July 2010

Abstract: The peptides of the transforming growth factor‐β (TGF‐β) family transduce their signal through ligand‐induced heteromeric complexes that consist of type I and type II serine/threonine kinases. Both TGF‐β receptors are abundant in many peripheral tissues, but clear evidence of their expression in cortical astrocytes and neurons has not been published so far. In this study, we investigated the expression of type I and type II TGF‐β receptors and their potential ligands (TGF‐β1, TGF‐β2, and TGF‐β3) in the CNS by using RT‐PCR and immunohistochemistry. Moreover, to further the study of those cell types that exhibit TGF‐β isoforms and related receptors, we examined through the use of RT‐PCR whether cortical neurons and astrocytes in culture express the mRNAs for TGF‐βs and their receptors. We show that the three TGF‐β isoform mRNAs are present in the CNS. However, although astrocytes in culture display all three isoforms, neurons in culture express only TGF‐β2. We have demonstrated that both type I and type II TGF‐β receptor mRNAs and proteins are present in the CNS and in cultures of cortical neurons and astrocytes. Thus, TGF‐βs may act as autocrine and paracrine signals in the CNS between both neurons and astrocytes via the same receptor systems as those found in peripheral tissues. TGF‐β1 has been shown to be induced following hypoxic‐ischemic brain injury and may play a critical role in the pathophysiology of degenerative processes in the CNS. In the present investigation, we confirmed that the expression of TGF‐β1 was increased markedly up until 24 h and thereafter was stable over the first 3 days following permanent occlusion of the middle cerebral artery in mice. However, whereas the expression of the type I TGF‐β receptor was not altered by the ischemic insult, the pattern of the type II TGF‐β receptors was modified dramatically in the ischemic area 3 days after the occlusion. These data show that, even if ligands are present, they may not be able to transduce their signal. Finally, the present study clearly demonstrates that a knowledge of the expression of ligand‐specific receptors following brain injury is a fundamental step in clarifying the involvement of cytokines in neurodegenerative diseases.
Bcl‐2 Protects Against Apoptosis in Neuronal Cell Line Caused by Thapsigargin‐Induced Depletion of Intracellular Calcium Stores
Authors: Wei Huafeng , Wei Wenlin , Bredesen Dale E. , Perry David C.
Published: July 2010

Abstract: The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca2+‐ATPase, was investigated in GT1‐7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca2+ ([Ca2+]i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca2+ diminished somewhat the size of the initial increase of [Ca2+]i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La3+ and by SKF 96365, a drug selective for receptor‐mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 µM BAPTA/AM, a cytosolic Ca2+ chelator, inhibited the peak [Ca2+]i caused by thapsigargin but did not inhibit the sustained elevation of [Ca2+]i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (“laddering”), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the proto‐oncogene bcl‐2 in GT1‐7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl‐2 did not alter either basal [Ca2+]i or the elevation of [Ca2+]i induced by thapsigargin. Our results suggest that abnormal Ca2+ release from endoplasmic reticulum caused by thapsigargin induces GT1‐7 death by apoptosis and that this effect does not depend on Ca2+ influx from the extracellular space. Bcl‐2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca2+ release in GT1‐7 neuronal cells.
The c‐erbAα Protooncogene Induces Apoptosis in Glial Cells via a Protein Kinase C‐ and bcl‐2‐Suppressible Mechanism
Authors: Llanos Susana , Caelles Carme , Azorín Inmaculada , Renau‐Piqueras Jaime , Fernández‐Luna José L. , Boscá Lisardo , Muñoz Alberto
Published: July 2010

Abstract: The c‐erbA protooncogene encodes the thyroid hormone (3,5,3′‐triiodothyronine; T3) receptor α1 (TRα1). c‐erbA/TRα1 is expressed in many cell types including glial cells, particularly in the immature state. We show here by morphological and biochemical criteria that c‐erbA induces apoptosis of glial B3.1 cells in serum‐deprived conditions. This effect is mostly T3 independent. Growth factors such as platelet‐derived growth factor, basic fibroblast growth factor, or transforming growth factor‐α prevent B3.1 + TRα1 cell death. Protein kinase C(PKC) activators also prevent the apoptosis phenomenon, an effect that was blocked by the PKC‐specific inhibitor GF109203X. Expression of an exogenous bcl‐2 gene led also to B3.1 + TRα1 cell survival. Neither a series of inhibitors including GF109203X nor T3 inhibits bcl‐2 action, indicating that bcl‐2 blocks a downstream step in the death‐promoting process. B3.1 + TRα1 cell apoptosis is not blocked by caspase‐1 or poly‐ADP‐ribosyltransferase inhibitors, suggesting that the activation of these classic pathways is not involved in the apoptotic mechanism. In addition, direct interaction with specific neuronal cells but not incubation with their conditioned medium inhibits also apoptosis of B3.1 + TRα1 cells. Our results show that c‐erbA promotes an apoptotic process in glial B3.1 cells that is suppressible by PKC activation and bcl‐2, probably by distinct mechanisms.
Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β‐Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture
Authors: Balázs Robert , Miller Stephan , Chun Yong , O'Toole John , Cotman Carl W.
Published: July 2010

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β‐adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum‐containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., (R,S)‐3,5‐dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG‐induced receptor down‐regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca2+]i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA‐AM or the PKC inhibitor Ro 31‐8220. An inhibitor of phospholipase C, U‐73122, markedly attenuated mGluR5‐activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist‐bound mGluR5 interacts with its coupled G protein.
Intraamniotic Ethyl Docosahexaenoate Administration Protects Fetal Rat Brain from Ischemic Stress
Authors: Glozman Sabina , Green Pnina , Yavin Ephraim
Published: July 2010

Abstract: Studies were conducted on the prenatal rat given a single intraamniotic injection of ethyl docosahexaenoate (Et‐DHA; 9.6–12 mmol per fetus) or subjected to an n‐3 fatty acid‐deficient diet to assess the role of docosahexaenoate on oxidative stress during episodes of ischemia. A time‐dependent decrease in the ability of brain slices from animals treated with Et‐DHA to produce thiobarbituric acid‐reactive substance (TBARS), most pronounced after 1 day (from 58.1 ± 4.22 to 15.9 ± 1.6 nmol/mg of DNA), was noticed on stimulation with Fe2+. Brain slices from fetuses treated for 1 day with Et‐DHA and those from untreated fetuses produced TBARS levels of 46.7 ± 6.5 and 114.8 ± 10.8 nmol/mg of DNA, respectively, after a 20‐min occlusion of the fetal‐maternal circulation at embryonic day 20, suggesting a protective effect of Et‐DHA. The protective effect of a single dose of Et‐DHA in utero remained high up to 3 days after injection (p < 0.001) and was long‐lasting, yet not significant, up to 3 days following birth. In agreement with a reduction in TBARS production by slices, the endogenous levels of TBARS in brains of Et‐DHA‐treated animals were lower than in the controls. Et‐DHA‐injected fetuses exhibited significantly higher levels of esterified DHA than the non‐injected controls. n‐3‐deficient diet given to dams for 2 weeks before birth did not affect the levels of TBARS production in control fetal brain slices but abolished the increase caused by ischemia. Et‐DHA administration for 24 h to n‐3‐deficient fetuses reduced the amount of TBARS produced by the fetal brain slices from 49.1 ± 8.5 to 31.7 ± 4.1 nmol/mg of DNA. A protective effect from oxidative damage after postischemic oxidative stress in fetal brain following DHA supplements is suggested, whereas the effect of n‐3 fatty acid deficiency in this regard is more ambiguous.
Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes
Authors: Sanna Enrico , Motzo Costantino , Usala Marcello , Pau Davide , Cagetti Elisabetta , Biggio Giovanni
Published: July 2010

Abstract: Expression of rat brain γ‐aminobutyric acid type A (GABAA) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABAA receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABAA receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage‐clamp conditions. The maximal amplitude of GABA‐induced Cl− currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half‐maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA‐induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ∼65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half‐maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up‐regulation of nigral GABAA receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β‐carbolines.