bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐Term Expression of Fos‐Related Antigen and Transient Expression of ΔFosB Associated with Seizures in the Rat Hippocampus and Striatum
Authors: Bing Guoying , Wang Wen , Qi Qiping , Feng Zhehui , Hudson Pearlie , Jin Lei , Zhang Wanqin , Bing Renjie , Hong Jau‐Shyong
Published: July 2010

Abstract: Systemic administration of kainic acid (KA), an analogue of glutamic acid, causes limbic seizures and pathophysiological changes in adult rats that are very similar to human temporal lobe epilepsy. One of the earliest changes in gene expression after treatment with KA is the induction of immediate‐early genes. The fos and jun families are frequently studied immediate‐early genes that are induced by KA. Several groups, including ours, have recently reported that a 35‐kDa Fos‐related antigen (FRA) is induced for a protracted time by various stimuli. It has been suggested that this FRA is ΔFosB, which has a molecular mass of ∼35 kDa. The present study characterizes the long‐term expression of FRA and ΔFosB after systemic treatment with KA. Immunocytochemistry and western blot analysis using an antibody that cross‐reacts with all known FRAs showed that a 35‐kDa FRA was induced at high levels in both the hippocampus and striatum for up to 1 month by KA. A semi‐quantitative PCR analysis showed that ΔFosB was induced by KA, but its expression lasted for only 6 h. This result was also verified by northern blot analysis. These results suggested that the 35‐kDa FRA with long‐term elevated levels seen with western blot analysis and immunocytochemistry is a new species of the FRA and not ΔFosB. The long‐term expression of FRA in both the hippocampus and striatum may be associated with the pathophysiological changes after KA administration.
Neuroprotective Effect of Somatostatin on Nonapoptotic NMDA‐Induced Neuronal Death: Role of Cyclic GMP
Authors: Forloni Gianluigi , Lucca Elisa , Angeretti Nadia , Chiesa Roberto , Vezzani Annamaria
Published: July 2010

Abstract: Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA‐induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7–10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5–10 µM), simultaneously applied with excitatory amino acid, significantly reduced in a dose‐dependent manner the neurotoxic effect of NMDA but not that of KA (0.25–0.5 mM). GABA (10 µM) partially protected neurons to a similar extent from NMDA‐ or KA‐induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201‐995; 10 µM) and vapreotide (RC 160; 10 µM), did not influence the NMDA‐dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl‐cyclic GMP (10 µM), a cyclic GMP analogue, whereas 8‐(4‐chlorphenylthio)‐cyclic AMP (10 µM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose‐dependent manner by SRIF (2.5–10 µM). Finally, both specific (Rp‐8‐bromoguanosine 3′,5′‐monophosphate, 10 µM) and nonspecific [1‐(5 isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), 10 µM] cyclic GMP‐dependent protein kinase (cGMP‐PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA‐induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP‐PK presumably by regulation of the intracellular Ca2+ level.
Nociceptin/Orphanin FQ Metabolism: Role of Aminopeptidase and Endopeptidase 24.15
Authors: Montiel Jose‐Luis , Cornille Fabrice , Roques Bernard P. , Noble Florence
Published: July 2010

Abstract: The endogenous opioid receptor‐like1 (ORL1) ligand, nociceptin/orphanin FQ (FGGFTGARKSARKLANQ), a heptadecapeptide structurally resembling dynorphin A, has recently been identified. The wide distribution of ORL1 mRNA and nociceptin/orphanin FQ precursor in the CNS, particularly in the limbic system regions and in several areas known to be involved in pain perception, suggests that nociceptin/orphanin FQ is potentially endowed with various central functions. In general, activation and/or inactivation of regulatory peptides occur through the action of cell surface peptidases. The physiological mechanisms under which nociceptin/orphanin FQ is metabolized should lead to a better understanding of its physiological functions. Mouse brain cortical slices were incubated in medium containing the heptadecapeptide in the presence or in the absence of peptidase inhibitors. The critical sites of enzymatic cleavage are Phe1‐Gly2, Ala7‐Arg8, Ala11‐Arg12, and Arg12‐Lys13 bonds. The major role played by metallopeptidases was confirmed by the complete protection of metabolism in the presence of EDTA. Aminopeptidase N and endopeptidase 24.15 are the two main enzymes involved in nociceptin/orphanin FQ metabolism, whereas endopeptidase 24.11 (involved in enkephalin [YGGFM(L)] catabolism) does not appear critically involved in nociceptin/orphanin FQ metabolism. The physiological relevance of aminopeptidase N and endopeptidase 24.15 in the heptadecapeptide metabolism remains to be determined.
Corticosteroid Modulation of Signal Transduction in the CATH.a Cell Line
Authors: Bundey Richard A. , Iredale Philip A. , Kendall David A.
Published: July 2010

Abstract: Noradrenergic neuronal networks originating in the locus coeruleus have been implicated in the stress response. In order to study this system in vitro, we have employed a locus coeruleus‐like cell line, CATH.a, and have determined the effect of dexamethasone on receptor‐mediated second messenger responses. The CATH.a cell line produced increases in intracellular cyclic AMP conversion in response to corticotrophin‐releasing factor (EC50 = 6.93 ± 1.26 nM, maximum conversion = 4.11 ± 0.20%) and vasoactive intestinal polypeptide (EC50 = 240 ± 40 nM, maximum conversion = 8.92 ± 1.24%). Forskolin (10 µM) increased conversion from 0.48 ± 0.05 to 6.39 ± 0.38%. The α2‐adrenoceptor agonist 5‐bromo‐N‐(4,5‐dihydro‐1H‐imidazol‐2‐yl)‐6‐quinoxalinamine (UK14304) inhibited the forskolin response with an IC50 of 6.76 ± 0.11 nM. Carbachol increased total 3H‐labelled inositol phosphate accumulation to a maximum of 3.01 ± 0.79 fold basal (EC50 = 7.94 ± 0.14 µM). Bradykinin produced a maximum 1.81 ± 0.05 fold basal stimulation of phosphoinositide hydrolysis (EC50 = 9.12 ± 0.16 nM). Both carbachol and bradykinin increased intracellular Ca2+ concentration probably via a combination of mobilisation of intracellular stores and gating of extracellular Ca2+. Incubation for 24 h with the glucocorticoid receptor agonist, dexamethasone (1 µM), significantly potentiated the receptor‐mediated phosphoinositide responses to all the agents tested; however, of the receptor‐mediated increases in cyclic AMP conversion, only the vasoactive intestinal polypeptide response was potentiated. These results show that the CATH.a cell line displays some of the properties expected of locus coeruleus neurons and that glucocorticoid receptor stimulation selectively modulates receptor‐mediated increases in second messenger formation.
Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin‐Deficient (mld) Mouse Brain
Authors: Sun Yilong , Xu Jing , Barbarese Elisa , Carson John H.
Published: July 2010

Abstract: Myelin‐deficient (mld) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP‐positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10−6 per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant (mld/mld, scid/scid) and single mutant (mld/mld, +scid/+scid) mice, indicating that the scid factor is not required for MBP gene reactivation in mld. The significance of DNA rearrangement in mammalian development is discussed.
NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins
Authors: Wenzel Andreas , Fritschy Jean Marc , Mohler Hanns , Benke Dietmar
Published: July 2010

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit‐specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area‐specific rearrangement of NR2B subunit expression following birth.
A Globular, Not Asymmetric, Form of Acetylcholinesterase Is Expressed in Chick Motor Neurons: Down‐Regulation Toward Maturity and After Denervation
Authors: Tsim Karl W. K. , Choi Roy C. Y. , Dong Tina T. X. , Wan David C. C.
Published: July 2010

Abstract: In vertebrate neuromuscular junctions, the postsynaptic specializations include the accumulation of acetylcholinesterase (AChE) at the synaptic basal lamina and the muscle fiber. Several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti‐AChE catalytic subunit, anti‐butyrylcholinesterase (BuChE) catalytic subunit, and anti‐AChE collagenous tail monoclonal antibodies, we demonstrated that the motor neurons of chick spinal cord expressed AChE in vivo and the predominant AChE was the globular form of the enzyme. Neither asymmetric AChE nor BuChE was detected in the motor neurons. The molecular mass of AChE catalytic subunit in the motor neuron was ∼105 kDa, which was similar to that of the globular enzyme from low‐salt extracts of muscle; both of them were ∼5 kDa smaller than the asymmetric AChE from high‐salt extracts of muscle. The level of AChE expression in the motor neurons decreased, as found by immunochemical and enzymatic analysis, during the different stages of the chick's development and after nerve lesion. Thus, the AChE activity at the neuromuscular junctions that is contributed by the presynaptic motor neurons is primarily the globular, not the asymmetric, form of the enzyme, and these contributions decreased toward maturity and after denervation.
Differential Expression of Type I, II, and V Adenylyl Cyclase Gene in the Postnatal Developing Rat Brain
Authors: Matsuoka Isao , Suzuki Yosuke , Defer Nicole , Nakanishi Hironori , Hanoune Jacques
Published: July 2010

Abstract: The developmental changes in the expression of mRNA encoding three major brain adenylyl cyclase (AC; EC 4.6.1.1) subtypes, type I (AC1), II (AC2), and V (AC5), were examined by in situ hybridization in rat brain from neonate to adult. During the early postnatal stage, levels of AC1 transcripts were very high in the cerebral cortex, striatum, thalamus, brainstem, and inferior colliculus. Then, AC1 mRNA levels rapidly decreased to the levels observed in the adult brain. In contrast, AC1 transcripts were very low at the early postnatal stage in the cerebellum and hippocampus and markedly increased during the second postnatal week. AC2 mRNA was widely distributed in rat brain throughout the development, and levels did not vary with different ages of the animal. AC5 mRNA was expressed to a limited extent in the neonatal brain, but levels dramatically increased during the second postnatal week in restricted regions, including the striatum, nucleus accumbens, and olfactory tubercle. The developing profiles of three AC gene transcripts were confirmed by northern blot analyses with mRNA isolated from different brain regions at different postnatal stages. In addition, the basal and forskolin‐, GTPγS‐, or Ca2+/calmodulin‐stimulated AC activity in plasma membrane preparations obtained from different brain regions at different ages were correlated with the age‐dependent changes in the region‐specific AC mRNA levels. These results demonstrate that different AC subtypes are expressed in the developing rat brain in a region‐ and age‐specific manner, suggesting specific roles not only in the synaptic transmission but also in the differentiation and maturation of neuronal cells in the developing brain.
Opioid Effects on 45Ca2+ Uptake and Glutamate Release in Rat Cerebral Cortex in Primary Culture
Authors: Vlaskovska Mila , Schramm Michael , Nylander Ingrid , Kasakov Lubomir , You Zhi‐Bing , Herrera‐Marschitz Mario , Terenius Lars
Published: July 2010

Abstract: Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on 45Ca2+ uptake and glutamate release. 45Ca2+ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK‐801. K+ depolarization‐induced 45Ca2+ uptake was also reduced by MK‐801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the 45Ca2+ uptake induced by depolarization. Opioid agonists, selective for μ‐, κ‐, and δ‐receptors, inhibited the 45Ca2+ uptake induced by K+ depolarization. The combination of low concentrations of MK‐801 and opioid agonists resulted in additive inhibition of K+‐ induced 45Ca2+ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca2+ uptake under acute, and perhaps also chronic, opiate treatment.
Ethanol Promotes Apoptosis in Cerebellar Granule Cells by Inhibiting the Trophic Effect of NMDA
Authors: Bhave Sanjiv V. , Hoffman Paula L.
Published: July 2010

Abstract: When primary cultures of cerebellar granule neurons are grown in a physiological concentration of KCl (5 mM) they undergo apoptosis, which can be prevented by growing the cells in the presence of N‐methyl‐d‐aspartate (NMDA). We now show that ethanol inhibits this trophic effect of NMDA, i.e., promotes apoptosis, and also inhibits the NMDA‐induced increase in intracellular Ca2+ concentration in cells grown in 5 mM KCl. Both effects of ethanol show a similar concentration dependence and are reversed by a high concentration of glycine, the co‐agonist at the NMDA receptor. The data suggest that the effect of ethanol on apoptosis is mediated, at least in part, by inhibition of NMDA receptor function. This effect of ethanol to increase apoptosis could contribute to the previously described in vivo sensitivity of the developing cerebellum to ethanol‐induced damage.
Prostaglandin E2 Induces Interleukin‐6 Synthesis in Human Astrocytoma Cells
Authors: Fiebich Bernd L. , Hüll Michael , Lieb Klaus , Gyufko Karin , Berger Mathias , Bauer Joachim
Published: July 2010

Abstract: Prostaglandins (PGs) and cytokines, such as interleukin‐1 (IL‐1) and interleukin‐6 (IL‐6), have been implicated in the etiopathology of various inflammatory and degenerative disorders, including Alzheimer's disease (AD) and prion diseases. Nonsteroidal antiinflammatory drugs (NSAIDs), potent inhibitors of PG synthesis, appear to be beneficial in the treatment of AD. To assess whether PGs are able to induce IL‐6 synthesis in cells of the CNS, IL‐6 mRNA and protein syntheses were measured in a human astrocytoma cell line after stimulation with different PGs. PGE1 and PGE2, but not PGD2 and PGF2α, led to a rapid and transient induction of IL‐6 mRNA, followed by IL‐6 protein synthesis. Furthermore, PGE2 potentiated IL‐1β‐induced IL‐6 mRNA synthesis. These results are discussed with respect to the participation of PGs in neurodegenerative diseases (and its inhibition by NSAIDs) by affecting cytokine expression.
Increased Expression of Cathepsins E and D in Neurons of the Aged Rat Brain and Their Colocalization with Lipofuscin and Carboxy‐Terminal Fragments of Alzheimer Amyloid Precursor Protein
Authors: Nakanishi Hiroshi , Amano Tutomu , Sastradipura Dewi F. , Yoshimine Yoshito , Tsukuba Takayuki , Tanabe Kazunari , Hirotsu Ichirou , Ohono Tomochika , Yamamoto Kenji
Published: July 2010

Abstract: Age‐related changes in the expression and localization of two distinct intracellular aspartic proteinases, cathepsin E (CE) and cathepsin D (CD), were investigated in the rat cerebral cortex and the brainstem by immunocytochemical and quantitative methods using discriminative antibodies specific for each enzyme. Non‐lysosomal CE was barely detectable in these two brain tissues in the embryonic stages, whereas relatively high expression of lysosomal CD was observed in embryonic tissues. After birth, CE was increasingly expressed in these tissues with aging to attain maximal levels at 30 months of age. Western blot analyses revealed that CE existed predominantly as the mature enzyme at 2 and 17 months of age, whereas it was present as not only the mature enzyme but also the proenzyme at 30 months of age. On the other hand, CD was mainly present in the mature form throughout development, although its level in these tissues was also significantly increased with aging. The CE‐positive cortical and brainstem neurons of the aged rat corresponded well with cells emitting autofluorescence for lipopigments. By the double‐staining technique, most of the CE‐positive cortical and brainstem neurons of the aged rat were also positive for antibody to the carboxyl‐terminal fragments of amyloid precursor protein (APP634–695), intracellular accumulation of which is thought to be associated with age‐related changes in the endosome/lysosome system. It is important that electron microscopy revealed that CE in brainstem neurons of the aged rat colocalized with CD in the lipofuscin‐containing lysosomes. These results indicate that aging results in the increased expression and lysosomal localization of CE in cortical and brainstem neurons and changes in the endosomal/lysosomal proteolytic system, which may be related to lipofuscinogenesis and altered intracellular APP metabolism.
Biochemical and Temporal Analysis of Events Associated with Apoptosis Induced by Lowering the Extracellular Potassium Concentration in Mouse Cerebellar Granule Neurons
Authors: Nardi Nurit , Avidan Galya , Daily Dvorah , Zilkha‐Falb Rina , Barzilai Ari
Published: July 2010

Abstract: We analyzed biochemically and temporally the molecular events that occur in the programmed cell death of mouse cerebellar granule neurons deprived of high potassium levels. An hour after switching the neurons to a low extracellular K+ concentration ([K+]o), a significant part of the genomic DNA was already cleaved to high‐molecular‐weight fragments. This phenomenon was intensified with the progression of the death process. Addition of cycloheximide to the neurons 4 h after high [K+]o deprivation resulted in no cell loss and complete recovery of the damaged DNA. DNA margination and nuclear fragmentation as assessed by 4,6‐diaminodiphenyl‐2‐phenylindole staining were observable in a few cells beginning ∼4 h after the removal of high [K+]o and developed to nuclear condensation 4 h later. Six hours after high [K+]o deprivation, the DNA was fragmented into oligonucleosome‐sized fragments. Within 6 h after removal of the extracellular K+, 50% of the neurons were committed to die and lost their ability to be rescued by readministration of 25 mM [K+]o. Similar to high [K+]o deprivation, inhibition of RNA or protein synthesis failed to halt neuronal degeneration of a similar percentage of cells 6 h after the onset of the death process. Mitochondrial function steadily decreased after [K+]o removal. An ∼40% decrease in RNA and protein synthesis was detected by 6 h of [K+]o removal during the period of cell death commitment; rates continued to decline gradually thereafter. The temporal characteristics of the DNA damage and recovery, DNA cleavage to oligonucleosome‐sized fragments, and the reduction in mitochondrial activity—events that occurred within the critical time—may indicate that these processes have an important part in the mechanism that committed the neurons to die.
Effect of Apolipoprotein E on Neurite Outgrowth and β‐Amyloid‐Induced Toxicity in Developing Rat Primary Hippocampal Cultures
Authors: Puttfarcken Pamela S. , Manelli Arlene M. , Falduto Michael T. , Getz Godfrey S. , LaDu Mary Jo
Published: July 2010

Abstract: The correlation between the ε4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established. However, the role of apoE in normal as well as pathological brain processes remains unclear. We evaluated the effect of apoE treatment on development and β‐amyloid (Aβ)‐induced toxicity using primary cultures of developing rat hippocampal neurons. The source of apoE was conditioned media from HEK cells stably transfected with human apoE3 or apoE4 cDNA, a preparation where apoE is lipid‐associated. Morphological and biochemical changes in the cultures were assessed at 1 and 3 days following low‐ and high‐density plating with either apoE3 or E4 with or without Aβ. Both apoE isoforms were neurotrophic, as measured by increased neurite length. Aged Aβ(1–42), a peptide preparation exhibiting extensive fibril and aggregate formation, is toxic to these cultures. Addition of apoE3 and E4 significantly and comparably attenuated the Aβ‐induced reduction in both neurite length and cell viability. The level of protection against this toxicity was proportional to the neurotrophic actions of the two apoE isoforms. Thus, apoE acts as a potent growth factor in both the absence and the presence of Aβ, supporting a potentially important role for apoE in neurobiology.
Acetylcholine Release in the Rat Prefrontal Cortex In Vivo: Modulation by α2‐Adrenoceptor Agonists and Antagonists
Authors: Tellez Stéphane , Colpaert Francis , Marien Marc
Published: July 2010

Abstract: We have previously shown that the release of acetylcholine (ACh) in the medial prefrontal cortex of the conscious rat, as measured by microdialysis, is increased following intraperitoneal injection of the selective α2‐adrenoceptor antagonist (+)‐efaroxan. To characterize further the receptor pharmacology of this response, the effects of other selective α2‐adrenoceptor ligands were examined. The α2‐adrenoceptor antagonists idazoxan (2.5 and 20 mg/kg), atipamezole (2.5 mg/kg), and fluparoxan (10 mg/kg) increased ACh outflow by up to 250–325% of basal levels over a 3‐h period following intraperitoneal injection. The α2‐adrenoceptor agonists UK‐14304 (2.5 mg/kg) and guanabenz (2.5 mg/kg) reduced ACh outflow by 80 and 60%, respectively. Clonidine (0.00063–0.16 mg/kg) had no significant depressant effect and at 2.5 mg/kg increased ACh outflow to 233% of basal levels. These results indicate a modulatory role for α2‐adrenoceptors on the release of ACh in the rat prefrontal cortex in vivo. Based on the facilitatory effects produced by the antagonists alone, this α2‐adrenoceptor modulation appears to be tonic and inhibitory. The ability of α2‐adrenoceptor antagonists to enhance ACh outflow suggests a therapeutic usefulness in disorders where cortical ACh release deficits have been implicated.
Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain
Authors: Morishita Rika , Saga Shinsuke , Kawamura Noriko , Hashizume Yoshio , Inagaki Toshiaki , Kato Kanefusa , Asano Tomiko
Published: July 2010

Abstract: The localization of two forms of the γ subunit of G proteins, γ3 and γ12, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ3 tended to decrease with age, whereas that of γ12 in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ3 was abundant in the neuropil, whereas γ12 was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ12‐positive cells, as well as levels of glial fibrillary acidic protein‐ and vimentin‐positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ12‐positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ12 is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ12 was predominant in glioma cells, such as C6 and GA‐1 cells, in contrast with the specific localization of γ3 in PC12 pheochromocytoma cells, which are neuron‐like cells. Taken together, the results indicate that γ3 and γ12 are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ3 and γ12 in the brain are related to the numbers and/or extent of maturation of these cells.
Lysophosphatidic Acid Increases Tight Junction Permeability in Cultured Brain Endothelial Cells
Authors: Schulze Charlotte , Smales Caroline , Rubin Lee L. , Staddon James M.
Published: July 2010

Abstract: Brain capillary endothelial cells are coupled by a continuous belt of complex high‐electrical‐resistance tight junctions that are largely responsible for the blood‐brain barrier. We have investigated mechanisms regulating tight junction permeability in brain endothelial cells cultured to maintain high‐resistance junctions. The phospholipid lysophosphatidic acid (LPA) was found to cause a rapid, reversible, and dose‐dependent decrease in transcellular electrical resistance in brain endothelial cells. LPA also increased the paracellular flux of sucrose, which, together with the resistance decrease, indicated increased tight junction permeability. Activation of protein kinase C attenuated the effect of LPA, suggesting that it was mediated by activation of a signalling pathway. LPA did not cause any obvious relocalization of adherens junction‐ or tight junction‐associated proteins. However, it did stimulate the formation of stress fibres, the recruitment of focal adhesion components, and the appearance of tyrosine phosphorylated protein at focal contacts. Our study shows that LPA is a modulator of tight junction permeability in brain endothelial cells in culture and raises the possibility that it triggers blood‐brain barrier permeability changes under (patho)physiological conditions.
δ‐Glutamate Receptors Are Differentially Distributed at Parallel and Climbing Fiber Synapses on Purkinje Cells
Authors: Zhao Hui‐Min , Wenthold Robert J. , Wang Ya‐Xian , Petralia Ronald S.
Published: July 2010

Abstract: Neurons containing multiple excitatory inputs may sort and target glutamate receptor subtypes to subsets of synapses. A good model for testing this hypothesis is the Purkinje cell, which expresses significant levels of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate, kainate, N‐methyl‐d‐aspartate, δ‐, and metabotropic glutamate receptors. Purkinje cells receive two excitatory inputs, the parallel and climbing fibers; the combined effect of stimulation of these two inputs is to produce long‐term depression of parallel fiber/Purkinje cell neurotransmission. Distribution of glutamate receptors in these two synapse populations in rat cerebella was studied using preembedding immunocytochemistry with antibodies to GluR1, GluR2/3, GluR5‐7, NR1, δ1/2, and mGluR1α. Moderate/dense postsynaptic staining was most frequent in postsynaptic densities and spines of both parallel and climbing fiber synapses with mGluR1α antibody, was intermediate in frequency with GluR2/3 and GluR5‐7 antibodies, and was least frequent with GluR1 and NR1 antibodies. The most striking finding was the absence of significant postsynaptic staining with δ1/2 antibody in climbing fiber synapses in adult animals, even though postsynaptic staining was prevalent in parallel fiber synapses with this antibody. In contrast to adults, moderate/dense postsynaptic immunolabeling of climbing fiber synapses with δ1/2 antibody was common in rats at 10 days postnatal. This study provides direct morphological evidence that δ‐glutamate receptors are differentially targeted to synapse populations. Our results support previous suggestions that δ2 is involved in development of parallel and climbing fiber synapses and in long‐term depression of parallel fiber/Purkinje synaptic responses in adults.
α1D L‐Type Ca2+‐Channel Currents: Inhibition by a β‐Adrenergic Agonist and Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) in Rat Pinealocytes
Authors: Chik Constance L. , Liu Qin‐Yue , Li Bing , Klein David C. , Zylka Mark , Kim Dong Sun , Chin Hemin , Karpinski Edward , Ho Anthony K.
Published: July 2010

Abstract: In this study the subunits of the dihydropyridine‐sensitive L‐type Ca2+ channels (L‐channels) expressed in rat pinealocytes were characterized using reverse transcription (RT)‐PCR analysis, and the modulation of these channels by adrenergic agonists and by pituitary adenylate cyclase‐activating polypeptide (PACAP) was studied using the patch‐clamp technique. RT‐PCR analysis showed that rat pinealocytes expressed α1D, α2b, β2, and β4 Ca2+‐channel subunit mRNAs. Other α1 subunit transcripts were either not expressed or present at very low levels, indicating that the pinealocytes express predominantly α1D L‐channels. Electrophysiological studies confirmed that the pineal expressed a single population of L‐channels. The L‐channel currents were inhibited by two agonists that elevate cyclic AMP: the β‐adrenergic agonist isoproterenol and PACAP. Similar inhibition was observed with a cyclic AMP analogue, 8‐bromo‐cyclic AMP. The presence of a cyclic AMP antagonist, Rp‐adenosine 3′,5′‐cyclic monophosphorothioate, blocked the inhibition by isoproterenol and PACAP. Norepinephrine, a mixed α‐ and β‐adrenergic agonist, also inhibited the L‐channel currents, but the inhibition was smaller. The smaller inhibition by norepinephrine was secondary to the simultaneous activation of α‐ and β‐adrenergic receptors. These results indicate that (a) pinealocytes express predominantly α1D L‐channels, and (b) the β‐adrenergic agonist isoproterenol and PACAP inhibit the L‐channel currents through elevation of cyclic AMP. However, an α‐adrenergic‐mediated mechanism also appears to be involved in the effect of norepinephrine on the L‐channel currents.
Transmitter‐Induced Calcium Responses Differ in Astrocytes Acutely Isolated from Rat Brain and in Culture
Authors: Kimelberg H. K. , Cai Z. , Rastogi P. , Charniga C. J. , Goderie S. , Dave V. , Jalonen T. O.
Published: July 2010

Abstract: Glial fibrillary acid protein (GFAP)‐positive astrocytes isolated from the cerebral cortices of 3–10‐day‐old rats frequently showed increased intracellular Ca2+ concentration responses to l‐glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonin [5‐hydroxytryptamine (5‐HT)]. The same cell that failed to respond to ATP or 5‐HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca2+ responses to glutamate but increased the responses to ATP and induced responses to 5‐HT. In primary cultures prepared from the cerebral cortices of 1‐day‐old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5‐HT. The lack of, or limited response to, 5‐HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAP‐negative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca2+ responses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5‐HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.
125I‐Galanin Binding Sites in Alzheimer's Disease: Increases in Hippocampal Subfields and a Decrease in the Caudate Nucleus
Authors: Rodríguez‐Puertas Rafael , Nilsson Siv , Pascual Julio , Pazos Angel , Hökfelt Tomas
Published: July 2010

Abstract: Using iodinated human galanin and autoradiography, galanin binding sites were studied in cortical and hippocampal areas and in some brainstem nuclei in the brains of eight patients with senile dementia of the Alzheimer type (SDAT) and of nine matched control cases. The highest density of binding sites was found in the substantia nigra with a less intense labeling in the hippocampus and cortical regions. In the SDAT cases a significant increase in number of galanin binding sites was found in some hippocampal areas, a decrease in the caudate nucleus, and no significant changes in frontal and entorhinal cortices. These findings suggest that some central galanin systems may be deranged in SDAT.
Cyclosporin A, an Immunosuppressive Drug, Induces Programmed Cell Death in Rat C6 Glioma Cells by a Mechanism that Involves the AP‐1 Transcription Factor
Authors: Mosieniak Grazyna , Figiel Izabela , Kaminska Bozena
Published: July 2010

Abstract: Cyclosporin A (CsA) is a clinically important immunosuppressive drug widely used to prevent graft rejection following organ or bone marrow transplantation. Although there are reports of serious neurologic alterations associated with the use of the drug, the precise mechanism of its action on the CNS still remains unknown. We studied the effects of CsA on the growth of C6 glioma cells. We found that CsA inhibits the growth of C6 glioma cells in a dose‐dependent manner and induces morphological changes such as shrinkage of the cell body and loss of extensions followed by cell death. The analysis of DNA from CsA‐treated cells revealed a ladder‐like pattern of fragmented DNA. Acridine orange staining showed the occurrence of apoptotic changes in nuclear morphology. Apoptotic morphological alterations were prevented by the treatment with cycloheximide. Altogether, our findings suggest that the CsA‐induced cell death of C6 glioma cells bears all the features characteristic of programmed cell death. We also observed a significant increase in the DNA‐binding activity of AP‐1 during CsA‐induced apoptosis. The AP‐1 induction preceded the appearance of apoptotic, morphological changes and was accounted for by an increase in the expression of c‐Jun protein. The occurrence of increased levels of AP‐1 complex and c‐Jun protein during CsA‐induced programmed cell death suggests its involvement in the induction of apoptosis.
Glial Alkalinization Detected In Vivo by 1H‐15N Heteronuclear Multiple‐Quantum Coherence‐Transfer NMR in Severely Hyperammonemic Rat
Authors: Kanamori Keiko , Ross Brian D.
Published: July 2010

Abstract: Brain [5‐15N]glutamine amide protons were selectively observed in vivo by 1H‐15N heteronuclear multiple‐quantum coherence‐transfer NMR in spontaneously breathing, severely hyperammonemic rats during intravenous [15N]ammonium acetate infusion and the subsequent recovery period. The linewidth of brain [5‐15N]‐glutamine amide proton Hz increased from 36 ± 2 Hz at 3.4 h to 58 ± 6 Hz after 5.7 h of infusion, a net increase of 22 ± 6 Hz. Concomitantly, brain ammonia concentration increased from 1.7 to 3.5 ± 0.2 µmol/g and the rat progressed from grade III to grade IV encephalopathy. On recovery to grade III and decrease of brain ammonia concentration to 1.3 µmol/g, the linewidth returned to 37 ± 2 Hz. In aqueous solution, [5‐15N]glutamine amide proton Hz underwent a 17‐Hz linebroadening when pH was raised from 7.1 to 7.5 at 37°C, due to the increased rate of base‐catalyzed exchange with water proton. Hence, linebroadening is a sensitive measure of changing intracellular pH. The 22‐Hz linebroadening observed in vivo in severely hyperammonemic grade IV rats strongly suggests that the intracellular pH increases from 7.1 to about 7.4–7.5 in astrocytes where glutamine is synthesized and mainly stored. Probable mechanisms for the ammonia‐induced alkalinization and decreased intraglial buffering capacity, as well as implications of the result for pathogenesis of hepatic encephalopathy, are discussed.
Altered Calcium Homeostasis in Cells Transformed by Mitochondria from Individuals with Parkinson's Disease
Authors: Sheehan J. P. , Swerdlow R. H. , Parker W. D. , Miller S. W. , Davis R. E. , Tuttle J. B.
Published: July 2010

Abstract: Parkinson's disease may be linked to defects in mitochondrial function. Mitochondrially transformed cells (cybrids) were created from Parkinson's disease patients or disease‐free controls. Parkinson's disease cybrids had 26% less complex I activity, but maintained comparable basal calcium and energy levels. Parkinson's disease cybrids recovered from a carbachol‐induced increase in cytosolic calcium 53% more slowly than controls even with lanthanum and thapsigargin blockade. Inhibition of complex I with the Parkinson's disease‐inducing metabolite 1‐methyl‐4‐phenylpyridinium (MPP+) similarly reduced the rate of recovery after carbachol. This MPP+‐induced reduction in recovery rates was much more pronounced in control cybrids than in Parkinson's disease cybrids. Parkinson's disease cybrids had less carbonyl cyanide m‐chlorophenylhydrazone‐releasable calcium. Bypassing complex I with succinate partially restored Parkinson's disease cybrid, and MPP+ suppressed control cybrid recovery rates. The subtle alteration in calcium homeostasis of Parkinson's disease cybrids may reflect an increased susceptibility to cell death under circumstances not ordinarily toxic.
Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats
Authors: Yamada Kiyofumi , Nabeshima Toshitaka
Published: July 2010

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO2−) and nitrate (NO3−) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO2− and NO3− were decreased by the intraperitoneal injection of NG‐nitro‐l‐arginine methyl ester, an inhibitor of nitric oxide synthase, whereas NG‐nitro‐d‐arginine methyl ester had no effect. l‐Arginine by itself increased NO2− and NO3− levels and diminished the reduction of their levels caused by NG‐nitro‐l‐arginine methyl ester. Direct infusion of l‐glutamate, N‐methyl‐d‐aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO2− and/or NO3− levels. The effects of N‐methyl‐d‐aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l‐glutamate and N‐methyl‐d‐aspartate were inhibited by NG‐nitro‐l‐arginine methyl ester and (±)‐3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid (CPP), an N‐methyl‐d‐aspartate receptor antagonist. These results suggest that NO2− and NO3− levels may be related to nitric oxide production in vivo.
Four 5‐Hydroxytryptamine7 (5‐HT7) Receptor Isoforms in Human and Rat Produced by Alternative Splicing: Species Differences Due to Altered Intron‐Exon Organization
Authors: Heidmann Doris E. A. , Metcalf Mark A. , Kohen Ruth , Hamblin Mark W.
Published: July 2010

Abstract: The serotonin (5‐HT) 5‐HT7 receptor subtype is thought to mediate a number of physiological effects in mammalian brain and periphery. Previous studies suggested that alternative splicing might contribute to 5‐HT7 receptor diversity as well. We now report that alternative splicing in human and rat tissues produces four 5‐HT7 receptor isoforms that differ in their predicted C‐terminal intracellular tails. Human and rat partial 5‐HT7 cDNAs and intronic sequences were identified and compared. In rat tissues, three 5‐HT7 isoforms, here called 5‐HT7(a), 5‐HT7(b), and 5‐HT7(c), are found. Rat 5‐HT7(a) [448‐amino acid (aa)] and 5‐HT7(b) (435‐aa) forms arise from alternative splice donor sites. A third new isoform found in rat, 5‐HT7(c) (470‐aa), results from a retained exon cassette. Three 5‐HT7 mRNA isoforms were also identified in human tissues, where only one isoform was previously described. Two human isoforms represent 5‐HT7(a) and 5‐HT7(b) forms (445‐ and 432‐aa), but the third form does not correspond to 5‐HT7(c). Instead, it constitutes a distinct isoform, 5‐HT7(d) (479‐aa), resulting from retention of a separate exon cassette. 5‐HT7(d) transcripts are not present in rat because the 5‐HT7(d)‐specifying exon is absent from the rat 5‐HT7 gene. A frame‐shifting homologue of the rat 5‐HT7(c)‐specifying exon is present in the human gene but is not used in the human tissues examined. Tissue‐specific splicing differences are present in human between brain and spleen. These studies suggest that alternative splicing may contribute to diversity of 5‐HT7 receptor action and that the human and rat repertoires of 5‐HT7 splice variants are substantially different.
Molecular Cloning and Characterization of the Human VGF Promoter Region
Authors: Canu Nadia , Possenti Roberta , Rinaldi Anna Maria , Trani Eugenia , Levi Andrea
Published: July 2010

Abstract: The VGF gene encodes a secretory protein that is expressed in a cell type‐restricted pattern in neuroendocrine cells and is up‐regulated by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. Here we report the isolation and characterization of the 5′‐terminal region of the human VGF gene. In addition to a TATA box and a CCAAT box located at canonical distances from the transcription start site, the human VGF promoter contains several consensus sequences for different transcription factors, including a cyclic AMP response element and an AP‐1 element, several GC boxes, and sequences homologous to other neuronal promoters. Transient transfection analysis demonstrates that 2.3 kb of the 5′‐flanking sequence acts as a tissue‐specific promoter, efficiently used only by neuronal cells that express endogenous VGF. Deletion analysis reveals that a positive regulatory region is located between nucleotides −458 to −204. Negative cis‐acting elements that repress promoter activity in cell lines that do not normally express VGF are located between nucleotides −2,305 and −573 and between −458 and −204. The 5′‐flanking region of the human VGF gene confers responsiveness to NGF, cyclic AMP, and phorbol ester treatment.
Effect of Ethanol on Extracellular Dopamine in Rat Striatum by Direct Perfusion with Microdialysis
Authors: Yim Hyeon Joo , Schallert Timothy , Randall Patrick K. , Bungay Peter M. , Gonzales Rueben A.
Published: July 2010

Abstract: The concentration‐related effects of ethanol on extracellular dopamine (DA) in rat striatum were studied by direct perfusion through microdialysis probes in freely moving rats. Two sets of three ethanol concentrations were separately tested using a Latin square experimental design. Potassium stimulation with high potassium (50 mM) in artificial CSF (ACSF) preceding ethanol treatment confirmed the neuronal function of dopaminergic cells by increasing DA concentrations to 200–1,500% of basal levels. The perfusion with calcium‐free ACSF applied at the end of each experiment confirmed the calcium dependency of the basal levels of extracellular DA by decreasing basal DA levels by 70%. The striatal volume measurement to examine the possible brain damage by direct ethanol perfusion suggested that ethanol did not increase the damage caused by the probe implantation at any ethanol concentration tested in this study. The 30‐min direct perfusion of 510 and 860 mM ethanol resulted in a significant concentration‐related stimulatory effect on the extracellular DA concentration in rat striatum (510 mM, 29% increase, p < 0.05; 860 mM, 66% increase, p < 0.05). However, there was no significant effect of ethanol at low concentrations, ≤170 mM. Considering the effective ethanol concentration in tissue areas in which DA is sampled, the data suggest that concentrations of ethanol associated with moderate intoxication do not directly affect the extracellular concentration of DA in the striatum. Therefore, the systemic effects of ethanol on striatal DA found in previous studies may be caused by the interaction with sites other than the striatum.