bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Solubility of High‐Molecular‐Mass Neurofilament Subunit by Suppression of Dephosphorylation: Its Relation to Axonal Transport
Authors: Tsuda Morihiro , Tashiro Tomoko , Komiya Yoshiaki
Published: July 2010

Abstract: To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200‐kDa subunit (NF‐H) in the axon was soluble in the 1% Triton‐containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68‐kDa subunit (NF‐L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF‐H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP‐dependent protein kinase, which resulted in NF disassembly. The carboxy‐terminal phosphorylation state of NF‐H probed with the phosphorylation‐sensitive antibodies was also not directly related to solubility. The solubility of NF‐H did not differ along the nerve. In contrast, the solubility of l‐[35S]methionine‐labeled, transported NF‐H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF‐H solubility is positively correlated with the rate of NF transport.
Preferential Potentiation of the Effects of Serotonin Uptake Inhibitors by 5‐HT1A Receptor Antagonists in the Dorsal Raphe Pathway: Role of Somatodendritic Autoreceptors
Authors: Romero Luz , Artigas Francesc
Published: July 2010

Abstract: 5‐HT1A autoreceptor antagonists enhance the effects of antidepressants by preventing a negative feedback of serotonin (5‐HT) at somatodendritic level. The maximal elevations of extracellular concentration of 5‐HT (5‐HText) induced by the 5‐HT uptake inhibitor paroxetine in forebrain were potentiated by the 5‐HT1A antagonist WAY‐100635 (1 mg/kg s.c.) in a regionally dependent manner (striatum > frontal cortex > dorsal hippocampus). Paroxetine (3 mg/kg s.c.) decreased forebrain 5‐HText during local blockade of uptake. This reduction was greater in striatum and frontal cortex than in dorsal hippocampus and was counteracted by the local and systemic administration of WAY‐100635. The perfusion of 50 µmol/L citalopram in the dorsal or median raphe nucleus reduced 5‐HText in frontal cortex or dorsal hippocampus to 40 and 65% of baseline, respectively. The reduction of cortical 5‐HText induced by perfusion of citalopram in midbrain raphe was fully reversed by WAY‐100635 (1 mg/kg s.c.). Together, these data suggest that dorsal raphe neurons projecting to striatum and frontal cortex are more sensitive to self‐inhibition mediated by 5‐HT1A autoreceptors than median raphe neurons projecting to the hippocampus. Therefore, potentiation by 5‐HT1A antagonists occurs preferentially in forebrain areas innervated by serotonergic neurons of the dorsal raphe nucleus.
Heterogeneity of β‐Adrenoceptors in Guinea‐Pig Brain: Radioligand Binding and Cyclic Nucleotide Generation
Authors: Neil Karen E. , Hernández Félix , Kendall David A. , Alexander Stephen P. H.
Published: July 2010

Abstract: In this report, we have examined the radioligand binding and second messenger signalling characteristics of β‐adrenoceptors in the guinea‐pig brain. [125I]lodocyanopindolol ([125I]ICYP)‐labelled sites in the cerebellum and cerebral cortex were of similar densities (Bmax 34 and 24 fmol·mg−1) and affinities (KD 20 and 55 pM), respectively. Analysis of competition for [125I]ICYP binding in the cerebellum was compatible with the presence of a β2‐adrenoceptor. In this tissue, isoprenaline evoked a cyclic AMP stimulation, and also potentiated cyclic GMP accumulations evoked in the presence of a nitric oxide donor, consistent with mediation via a β2‐adrenoceptor. The [125I]ICYP binding profile in the cerebral cortex did not comply with those previously described for β‐adrenoceptor subtypes, and isoprenaline failed to alter significantly cyclic AMP accumulation in the cerebral cortex, hippocampus, or neostriatum, even in the presence of forskolin or a phosphodiesterase inhibitor. Isoprenaline was also without effect on cyclic GMP accumulation or phosphoinositide turnover in the cerebral cortex. These results suggest that the guinea‐pig cerebellum expresses a functional β2‐adrenoceptor coupled to cyclic AMP generation, and potentiation of cyclic GMP accumulation. However, the guinea‐pig cerebral cortex displays binding sites that exhibit β‐adrenoceptor‐like pharmacology but fail to show functional coupling to cyclic AMP, cyclic GMP, or phosphoinositide signalling systems.
Modulation of Serotonin Transporter Activity by a Protein Kinase C Activator and an Inhibitor of Type 1 and 2A Serine/Threonine Phosphatases
Authors: Sakai Norio , Sasaki Keiko , Nakashita Mayumi , Honda Shigeru , Ikegaki Natsu , Saito Naoaki
Published: July 2010

Abstract: We studied the effects of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), a protein kinase C (PKC) activator, and calyculin A (CLA), an inhibitor of type 1 and 2A serine/threonine phosphatases, on serotonin uptake by a human placenta choriocarcinoma cell line (BeWo) and COS‐7 cells expressing recombinant serotonin transporter (SET). In BeWo cells, treatment with TPA decreased imipramine‐sensitive serotonin uptake with a reduction in Vmax without affecting Km. CLA also decreased imipramine‐sensitive serotonin uptake in a manner similar to that of TPA. TPA and CLA also decreased the uptake activity of recombinant SET expressed in COS‐7 cells as seen in BeWo cells. These effects of TPA and CLA were reversed by staurosporine, a protein kinase inhibitor. To elucidate whether the inhibitory effects of TPA and CLA were due to direct phosphorylation of SET by PKC, site‐directed mutagenesis of five putative PKC phosphorylation sites in SET was performed. Serotonin uptake was also down‐regulated by TPA and CLA in all nine mutants, suggesting that these inhibitory modulation of SET activity did not act via direct phosphorylation of SET by PKC.
NMDA Receptor‐Mediated Regulation of AMPA Receptor Properties in Organotypic Hippocampal Slice Cultures
Authors: Gellerman David M. , Bi Xiaoning , Baudry Michel
Published: July 2010

Abstract: Activation of the calcium‐dependent protease calpain has been proposed to be a necessary step in the formation of long‐term potentiation (LTP) in the hippocampus, and stimulation of N‐methyl‐d‐aspartate (NMDA) receptors leads to an increase in intracellular calcium concentration, calpain activation, proteolysis of cytoskeletal elements, and modification of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor properties. In the present study, we evaluated the effects of NMDA treatment of cultured hippocampal slices on the properties of AMPA receptors. Cultured hippocampal slices were treated with NMDA (100 µM) for 15 min and [3H]AMPA binding to membrane fractions was measured. NMDA‐treated slices exhibited an increase in both “high‐affinity” and “low‐affinity” [3H]‐AMPA binding, with smaller changes in 6‐cyano‐7‐nitro[3H]quinoxaline‐2,3‐dione binding. The increase in [3H]AMPA binding was significantly reduced by preincubation of cultures with calpain inhibitor I or calpeptin (100 µM). Furthermore, NMDA exposure decreased the number of GluR1 subunits of AMPA receptors detected by an antibody against the C‐terminal domain of the subunit in western blots and resulted in the formation of a lower molecular weight species detected by an antibody against the N‐terminal domain. Both effects were completely prevented by calpain inhibitors. These results indicate that NMDA receptor activation produces calpain activation and complex modifications of AMPA receptor properties, which could be involved in NMDA receptor‐mediated changes in synaptic efficacy.
Acute and Chronic Treatments with Citalopram Lower Somatostatin Levels in Rat Brain Striatum Through Different Mechanisms
Authors: Prosperini Elena , Rizzi Massimo , Fumagalli Fabio , Tarizzo Gianluca , Samanin Rosario , Bendotti Caterina
Published: July 2010

Abstract: The suggestion that somatostatin is involved in the pathophysiology of obsessive‐compulsive disorder and the evidence that selective serotonin reuptake inhibitors show significant antiobsessional effect prompted us to examine the effect of citalopram, a selective and potent serotonin reuptake inhibitor, on the somatostatinergic system in different brain regions of the rat. A single intraperitoneal injection of 10 mg/kg citalopram significantly reduced somatostatin levels in the striatum and nucleus accumbens after 4 but not 1, 8, or 24 h. No changes were found in hippocampus. In addition, we found that the K+‐evoked overflow of somatostatin‐like immunoreactivity from striatal slices was significantly increased 1 h after a single injection of citalopram and was still higher, although not significantly, 4 h after the drug injection. Levels of preprosomatostatin mRNA were unchanged in striatum and accumbens 1 and 4 h after a single drug administration. In rats treated with citalopram (10 mg/kg i.p.) twice daily for 14 days, the levels of somatostatin and its mRNA were significantly decreased in the striatum but not in other brain regions 24 h after the last dose. No change was found in the basal or K+‐evoked overflow of somatostatin‐like immunoreactivity at 1, 4, and 24 h after the last drug injection. These results suggest that acute and chronic treatment with citalopram reduces somatostatin levels in striatum by different mechanisms. Whereas a single dose of the drug reduces somatostatin levels by increasing the release of the peptide, repeated drug treatment reduces the biosynthesis of somatostatin.
α2‐Macroglobulin as a β‐Amyloid Peptide‐Binding Plasma Protein
Authors: Du Yansheng , Ni Binhui , Glinn Michele , Dodel Richard C. , Bales Kelly R. , Zhang Zhiyuan , Hyslop Paul A. , Paul Steven M.
Published: July 2010

Abstract: The β‐amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α2‐macroglobulin (α2M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds 125I‐Aβ(1–42) with high affinity (apparent dissociation constant of 3.8 × 10−10M). Approximately 1 mol of Aβ is bound per mole of α2M. Both native and methylamine‐activated α2M bind 125I‐Aβ(1–42). The binding of 125I‐Aβ(1–42) to α2M is enhanced by micromolar concentrations of Zn2+ (but not Ca2+) and is inhibited by noniodinated Aβ(1–42) and Aβ(1–40) but not by the reverse peptide Aβ(40‐1) or the cytokines interleukin 1β or interleukin 2. α1‐Antichymotrypsin, another plaque‐associated protein, inhibits both the binding of 125I‐Aβ(1–42) to α2M as well as the degradation of 125I‐Aβ(1–42) by proteinase‐activated α2M. Moreover, the binding of 125I‐Aβ(1–42) to α2M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α2M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.
AP‐1 and Egr DNA‐Binding Activities Are Increased in Rat Brain During Ethanol Withdrawal
Authors: Beckmann Alison M. , Matsumoto Izuru , Wilce Peter A.
Published: July 2010

Abstract: The DNA‐binding activities of AP‐1 and Egr proteins were investigated in nuclear extracts of rat brain regions during ethanol withdrawal. Both DNA‐binding activities were transiently elevated in the hippocampus and cerebellum 16 h after withdrawal. In the cerebral cortex, AP‐1 and Egr DNA‐binding activities increased at 16 h and persisted until 32 and 72 h, respectively. The AP‐1 DNA‐binding activities in all regions at all times after withdrawal were composed of FosB, c‐Jun, JunB, and JunD. c‐Fos was detected at all times in the cerebral cortex, at 16 h only in the hippocampus, and from 16 to 72 h in the cerebellum. Withdrawal severity did not affect the composition of the AP‐1 DNA‐binding activities. Two Egr DNA‐binding activities were present in the cortex and hippocampus. The faster‐migrating complex predominated in hippocampus, and only the slower‐migrating complex (identified as Egr‐1) was present in the cerebellum. The increase in DNA‐binding activity of immediate early gene‐encoded transcription factors supports their proposed role in initiating a cascade of altered gene expression underlying the long‐term neuronal response to ethanol withdrawal.
Distribution of Ankyrin Isoforms and Their Proteolysis After Ischemia and Reperfusion in Rat Brain
Authors: Harada Kazuki , Fukuda Shiro , Kunimoto Manabu , Yoshida Ken‐ichi
Published: July 2010

Abstract: The distribution of brain‐type ankyrin (ankyrinB, 212 kDa) and erythrocyte‐type ankyrin (ankyrinR, 239 kDa) was investigated in the subcellular fractions of rat forebrain (P1, 1,000 g pellet; P2, 15,000 g pellet; P3, 100,000 g pellet; S, 100,000 g supernatant) by immunoblotting using specific antibodies. The P2 fraction contained ∼40% of the 212‐ and 163‐kDa isoforms of ankyrinB and the 239‐kDa isoform of ankyrinR. Further subfractionation of the P2 by Percoll gradient centrifugation followed by separation of myelin showed association of the three ankyrin isoforms with the synaptosome‐rich fraction but not with the myelin‐rich fraction. The plasma membrane‐rich P3 fraction contained a concentration of ankyrin isoforms similar to that in the P2 fraction. In vitro proteolysis of ankyrin in the P2 fraction with calpain showed that the 212‐kDa ankyrinB was more susceptible to calpain than was ankyrinR. In the two‐vessel occlusion model, ischemia for 30 min generated the 160‐kDa fragment of ankyrinR, and reperfusion for 60 min after 30 min of ischemia remarkably increased the 160‐kDa fragment. The reperfusion also significantly decreased the 212‐kDa isoform of ankyrinB. Both ischemia‐reperfusion and in vitro proteolysis with calpain generated the 160‐kDa fragment of ankyrinR, suggesting the involvement of calpain.
Aggregation State‐Dependent Activation of the Classical Complement Pathway by the Amyloid β Peptide
Authors: Webster Scott , Bradt Bonnie , Rogers Joseph , Cooper Neil
Published: July 2010

Abstract: Activation of the classical complement pathway has been widely investigated in recent years as a potential mechanism for the neuronal loss and neuritic dystrophy characteristic of Alzheimer's disease (AD) pathogenesis. We have previously shown that amyloid β peptide (Aβ) is a potent activator of complement, and recent evidence suggesting that the assembly state of Aβ is crucial to the progress of the disease prompted efforts to determine whether the ability of Aβ to activate the classical complement pathway is a function of the aggregation state of the peptide. In this report, we show that the fibrillar aggregation state of Aβ, as determined by thioflavin T fluorometry, electron microscopy, and staining with Congo red and thioflavine S, is precisely correlated with the ability of the peptide to induce the formation of activated fragments of the complement proteins C4 and C3. These results suggest that the classical complement pathway provides a mechanism whereby complement‐dependent processes may contribute to neuronal injury in the proximity of fibrillar but not diffuse Aβ deposits in the AD brain.
Characterization of 5,7‐Dichlorokynurenate‐Insensitive d‐[3H]Serine Binding to Synaptosomal Fraction Isolated from Rat Brain Tissues
Authors: Matoba Masaki , Tomita Urara , Nishikawa Toru
Published: July 2010

Abstract: To explore target sites for endogenous d‐serine that are different from the glycine site of the N‐methyl‐d‐aspartate (NMDA) type glutamate receptor, we have studied the binding of d‐[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 µM) of the glycine site antagonist 5,7‐dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled d‐serine. Association, dissociation, and saturation experiments indicated that d‐[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a KD of 614 nM and a Bmax of 2.07 pmol/mg of protein. d‐Serine, l‐serine, and glycine produced a total inhibition of the specific DCK‐insensitive d‐[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7‐chlorokynurenate failed to inhibit the binding at 10 µM. The profiles of displacement of the DCK‐insensitive d‐[3H]serine binding by various amino acids and glutamate and glycine receptor‐related compounds differ from those of any other defined recognition sites. DCK‐insensitive d‐[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK‐insensitive d‐[3H]serine binding site could be a novel candidate for a target for endogenous d‐serine in mammalian brains.
Subfamily of Olfactory Receptors Characterized by Unique Structural Features and Expression Patterns
Authors: Kubick Stefan , Strotmann Jörg , Andreini Isabella , Breer Heinz
Published: July 2010

Abstract: The complex chemospecificity of the olfactory system is probably due to the large family of short‐looped, heptahelical receptor proteins expressed in neurons widely distributed throughout one of the several zones within the nasal neuroepithelium. In this study, a subfamily of olfactory receptors has been identified that is characterized by distinct structural features as well as a unique expression pattern. Members of this receptor family are found in mammals, such as rodents and opossum, but not in lower vertebrates. All identified subtypes comprise an extended third extracellular loop that exhibits amphiphilic properties and contains numerous charged amino acids in conserved positions. Olfactory sensory neurons expressing these receptor types are segregated in small clusters on the tip of central turbinates, thus representing a novel pattern of expression for olfactory receptors. In mouse, genes encoding the new subfamily of receptors were found to be harbored within a small contiguous segment of genomic DNA. Based on species specificity as well as the unique structural properties and expression pattern, it is conceivable that the novel receptor subfamily may serve a special function in the olfactory system of mammals.
Mitochondrial Permeability Transition in the Central Nervous System: Induction by Calcium Cycling‐Dependent and ‐Independent Pathways
Authors: Kristal Bruce S. , Dubinsky Janet M.
Published: July 2010

Abstract: Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of isolated CNS mitochondria to elevated calcium or phosphate or both produced loss of absorbance indicative of mitochondrial swelling. The absorbance decreases were prevented by ADP and Mg2+ and reduced by cyclosporin A, dithiothreitol, and N‐ethylmaleimide. Ruthenium red prevented calcium cycling‐induced, but only attenuated phosphate‐induced losses of absorbance. In cultured astrocytes permeabilized with digitonin or treated with the calcium ionophore, 4‐bromo‐A23187, elevations of external calcium altered mitochondrial morphology visualized with the dye, JC‐1, from rod‐like to rounded, swollen structures. Similar changes were observed in digitonin‐permeabilized astrocytes exposed to phosphate. The incidence of calcium‐induced changes in astrocyte mitochondria was prevented by Mg2+ and pretreatment with dithiothreitol and N‐ethylmaleimide, and was reduced by cyclosporin A, ADP, and butacaine alone or in combinations. Ruthenium red and the Na+/Ca2+ exchange inhibitor CGP 37157 blocked calcium cycling and prevented mitochondrial shape changes in digitonin‐treated, but not ionophore‐treated astrocytes. Thus, the demonstrated induction conditions and pharmacological profile indicated the existence of an mPT in brain mitochondria. The mPT occurred consequent to activation of calcium cycling‐dependent and ‐independent pathways. Induction of an mPT could contribute to neuronal injury following ischemia and reperfusion.
Comparison Between the Timing of JNK activation, c‐Jun Phosphorylation, and Onset of Death Commitment in Sympathetic Neurones
Authors: Virdee Kanwar , Bannister Andrew J. , Hunt Stephen P. , Tolkovsky Aviva M.
Published: July 2010

Abstract: We have investigated the relationship between c‐Jun N‐terminal kinase (JNK) activity, apoptosis, and the potential of survival factors to rescue primary rat sympathetic neurones deprived of trophic support. Incubation of sympathetic neurones in the absence of nerve growth factor (NGF) caused a time‐dependent increase in JNK activity, which became apparent by 3 h and attained maximal levels that were three‐ to fourfold higher than activity measured in neurones maintained for the same periods with NGF. Continuous culture in the presence of either NGF or the cyclic AMP analogue 4‐(8‐chlorophenylthio) cyclic AMP (CPTcAMP) not only prevented JNK activation from occurring, but also suppressed JNK activity that had been elevated by prior culture of the neurones in the absence of trophic support. When either NGF or CPTcAMP was added to cultures that had been initially deprived of neurotrophic support for up to 10 h, this resulted in complete suppression of total JNK activity, arrest of apoptosis, and rescue of >90% of the neurones that did not display apoptotic morphology by this time. However, when either agent was added after more protracted periods of initial neurotrophin deprivation (≥ 14 h), although this also resulted in near‐complete suppression of total JNK activity and short‐term arrest of apoptosis, not all of the neurones that appeared to be nonapoptotic at the time of agent addition were rescued. The lack of death commitment after 10 h of maintained JNK activity was not due to a late induction of c‐Jun expression, because the majority of newly isolated sympathetic neurones had already been expressing high levels of c‐Jun in their nuclei for several hours, yet were capable of being rescued by NGF. Elevation of JNK activity as a result of neurotrophic‐factor deprivation was also associated with enhanced phosphorylation of c‐Jun, assessed by immunoblot analysis and immunocytochemistry, and addition of NGF to cultures previously deprived of neurotrophic support resulted in a reversion of the state of phospho‐c‐Jun to that observed in cultures that had been maintained in the continuous presence of trophic support. We conclude that activation of JNK and c‐Jun phosphorylation are not necessarily rate‐limiting for apoptosis induction. In some neurones undergoing prolonged NGF deprivation, suppression of JNK activity and c‐Jun dephosphorylation by NGF may be insufficient to effect their rescue. Thus, if c‐Jun mediates death by increasing the expression of “death” genes, these must become effective very close to the death commitment point.
Selective Changes in Cell Bodies and Growth Cones of Nerve Growth Factor‐Differentiated PC12 Cells Induced by Chemical Hypoxia
Authors: Gibson Gary , Toral‐Barza Lourdes , Zhang Hui
Published: July 2010

Abstract: Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca2+ in growth cones and cell bodies. Hypoxia increased [Ca2+]i and exaggerated its response to K+ depolarization in both parts of the cells. [Ca2+]i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K+ or hypoxia. Ca2+‐channel blockers selectively altered these responses. The L‐channel blocker nifedipine reduced [Ca2+]i following K+ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N‐channel blocker ω‐conotoxin GVIA (ω‐CgTX) diminished K+‐induced changes in [Ca2+]i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, ω‐CgTX diminished K+‐induced changes by 50–75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and ω‐CgTX diminished the [Ca2+]i response to K+ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca2+ entry with K+ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca2+ regulation in the growth cone and cell body of the same cell.
Eating‐Induced Dopamine Release from Mesolimbic Neurons Is Mediated by NMDA Receptors in the Ventral Tegmental Area: A Dual‐Probe Microdialysis Study
Authors: Westerink Ben H. C. , Kwint Henk‐Frans , De Vries Jan B.
Published: July 2010

Abstract: This study was aimed at identifying the neuronal pathways that mediate the eating‐induced increase in the release of dopamine in the nucleus accumbens of the rat brain. For that purpose, a microdialysis probe was implanted in the ventral tegmental area and a second probe was placed in the ipsilateral nucleus accumbens. Receptor‐specific compounds acting on GABAA (40 µM muscimol; 50 µM bicuculline), GABAB (50 µM baclofen), acetylcholine (50 µM carbachol), NMDA [30 µM (±)‐3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid (CPP)], and non‐NMDA [300 µM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX)] receptors were infused into the ventral tegmental area by retrograde dialysis, whereas extracellular dopamine was recorded in the ipsilateral nucleus accumbens. Intrategmental infusion of muscimol or baclofen decreased extracellular dopamine in the ipsilateral nucleus accumbens; CPP and CNQX were without effect, and bicuculline and carbachol increased dopamine release. During infusion of the various compounds, food‐deprived rats were allowed to eat for 10 min. The infusions of muscimol, bicuculline, baclofen, carbachol, and CNQX did not prevent the eating‐induced increase in extracellular dopamine in the nucleus accumbens. However, during intrategmental infusion of CPP, the eating‐induced increase in extracellular dopamine in the nucleus accumbens was suppressed. These results indicate that a glutamatergic projection to the ventral tegmental area mediates, via an NMDA receptor, the eating‐induced increase in dopamine release from mesolimbic dopamine neurons.
Metabotropic Glutamate Receptor Subtype mGluR1α Stimulates the Secretion of the Amyloid β‐Protein Precursor Ectodomain
Authors: Nitsch Roger M. , Deng Amy , Wurtman Richard J. , Growdon John H.
Published: July 2010

Abstract: To examine the effects of glutamatergic neurotransmission on amyloid processing, we stably expressed the metabotropic glutamate receptor subtype 1α (mGluR1α) in HEK 293 cells. Both glutamate and the selective metabotropic agonist 1‐amino‐1,3‐cyclopentanedicarboxylic acid (ACPD) rapidly increased phosphatidylinositol (PI) turnover four‐ to fivefold compared with control cells that were transfected with the expression vector alone. Increased PI turnover was effectively blocked by the metabotropic antagonist α‐methyl‐4‐carbophenylglycine (MCPG), indicating that heterologous expression of mGluR1α resulted in efficient coupling of the receptors to G protein and phospholipase C activation. Stimulation of mGluR1α with glutamate, quisqualate, or ACPD rapidly increased secretion of the APP ectodomain (APPs); these effects were blocked by MCPG. The metabotropic receptors were coupled to APP processing by protein kinases and by phospholipase A2 (PLA2), and melittin, a peptide that stimulates PLA2, potently increased APPs secretion. These data indicate that mGluR1α can be involved in the regulation of APP processing. Together with previous findings that muscarinic and serotonergic receptor subtypes can increase the secretion of the APP ectodomain, these observations support the concept that proteolytic processing of APP is under the control of several major neurotransmitters.
Cross Talk Between Substance P and Melittin‐Activated Cellular Signaling Pathways in Rat Lactotroph‐Enriched Cell Cultures
Authors: Mau Søren E. , Vilhardt Hans
Published: July 2010

Abstract: We have investigated the possible interaction (cross talk) between the phospholipase A2 (PLA2) and inositol 1,4,5‐trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph‐enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [3H]‐arachidonic acid ([3H]AA) from [3H]AA‐labeled enriched lactotrophs in a dose‐dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKCα and β immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)‐stimulated cells. Mellitin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA2 inhibitors quinacrine and aristolochic acid resulted in a dose‐dependent inhibition of melittin‐stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) dose‐dependently increased levels of [3H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [3H]AA formation. After long‐term treatment (24 h) of cells with TPA, the effect of TPA on [3H]AA production was not different from control, whereas SP still displayed [3H]AA‐releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP‐stimulated [3H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [3H]AA induced by SP, whereas PTX had no effect on SP‐stimulated generation of 3H‐inositol phosphates. On the basis of these results, it is concluded that (1) the PLA2 pathways interfere with the phosphoinositide‐PLC signaling system at the level of PKC isozymes α and β, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA2 pathway at a level at or beyond PLA2, and this effect is mediated, in part, through PKCα and β species and (for SP) intracellular Ca2+ recruited from internal stores as well as from external sources; and (3) SP also activates PLA2 through a PTX‐sensitive pathway distinct from the one coupled to phosphoinositide‐PLC, which is PTX insensitive.
Transfection of Reaggregating Embryonic Chicken Retinal Cells with an Antisense 5′‐DNA Butyrylcholinesterase Expression Vector Inhibits Proliferation and Alters Morphogenesis
Authors: Robitzki Andrea , Mack Alexandra , Chatonnet Arnaud , Layer Paul G.
Published: July 2010

Abstract: The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5′ upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate‐mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina‐like spheres, so‐called rosetted or stratified retinospheroids. We show that antisense 5′‐BChE gene expression decreased the steady‐state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5′‐BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.
Serum Opens Tight Junctions and Reduces ZO‐1 Protein in Retinal Epithelial Cells
Authors: Chang Chih‐wei , Wang Xiliang , Caldwell Ruth B.
Published: July 2010

Abstract: We have shown previously that serum inhibits tight junction formation in a retinal epithelial cell culture model for the blood‐brain barrier. We have now examined in detail the effects of serum on the tight junctions. Our data show that serum induces a breakdown in tight junction function as indicated by decreased transepithelial electrical resistance and increased permeability. Rat serum had effects similar to those of bovine serum, indicating that the activity is species‐independent. The effect is concentration‐dependent, reversible, and specific for the apical surface, suggesting the involvement of a specific receptor‐ligand interaction. Differences in the time course, response magnitude, and structural manifestations between the serum‐induced breakdown and that induced by switching the cultures to a low‐calcium medium suggest fundamental differences in their mechanisms. The calcium switch results in an immediate and complete junctional breakdown with cell retraction and perinuclear translocation of both actin and the tight junction protein zonula occludens‐1. The serum‐induced breakdown occurs slowly, is incomplete, and is manifested structurally by decreases in zonula occludens‐1 protein, whereas actin organization is unchanged. Thus, serum induces a specific breakdown in retinal epithelial cell tight junctions that may be mediated by effects on the expression of zonula occludens‐1.
Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain
Authors: Longuemare Maria C. , Swanson Raymond A.
Published: July 2010

Abstract: Elevated extracellular potassium concentration ([K+]e) has been shown to induce reversal of glial Na+‐dependent glutamate uptake in whole‐cell patch clamp preparations. It is uncertain, however, whether elevated [K+]e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K+]e (by equimolar substitution of K+ for Na+), and glutamate accumulation was measured by HPLC. With [K+]e elevations to 60 mM, medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100‐fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d‐aspartate, and parallel results were obtained; no increase in medium d‐aspartate content resulted from [K+]e elevation up to 90 mM, whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K+]e attainable in brain.
Structural Analysis of the Proximal Region of the Microtubule‐Associated Protein 1B Promoter
Authors: Liu Dong , Fischer Itzhak
Published: July 2010

Abstract: Microtubule‐associated protein 1B (MAP1B) is a major cytoskeletal protein expressed early during development of the nervous system. Previous analysis of the MAP1B gene has identified two alternative promoters that can independently regulate neuron‐specific expression of MAP1B. To further characterize the MAP1B promoters, we performed DNase I hypersensitivity assays in vivo over a range of 8.5 kb surrounding the transcription initiation sites. These studies identified a DNase I‐hypersensitive site that was present in brain but not liver nuclei at the proximal region of the MAP1B promoter, located between the two transcription initiation sites. Fine mapping by S1 nuclease sensitivity localized two adjacent sites in the proximal promoter region that contained three symmetrical inverted repeats. Electrophoresis mobility shift assays showed that proteins present in nuclear extracts can bind two consensus regulatory elements present within the proximal promoter region, Sp1 and cyclic AMP response element. In addition, there was a specific nuclear protein binding activity with two common sequences, a “neuronal motif” and a TCC repeat motif. This binding activity was much more abundant in liver than in brain nuclear extracts, suggesting that it may represent a negative control element in the tissue‐specific expression of the MAP1B gene.
Association of Brain Protein Phosphatase 1 with Cytoskeletal Targeting/Regulatory Subunits
Authors: Colbran Roger J. , Bass Martha A. , McNeill R. Blair , Bollen Mathieu , Zhao Sumin , Wadzinski Brian E. , Strack Stefan
Published: July 2010

Abstract: Protein phosphatase 1 catalytic subunit (PP1C) is highly enriched in isolated rat postsynaptic densities. Gel overlay analyses using digoxigenin (DIG)‐labeled PP1C revealed four major rat brain PP1C‐binding proteins (PP1bps) with molecular masses of ≈216, 175, 134, and 75 kDa, which were (1) more abundant in brain than other rat tissues; (2) differentially expressed in microdissected brain regions; and (3) enriched in isolated cortex postsynaptic densities. PP1bp175, PP1bp134, PP1bp75, and PP1C were partially released from forebrain particulate extracts by incubation at low ionic strength, which destabilizes the actin cytoskeleton. Size‐exclusion chromatography of solubilized extracts separated two main PP1 activities (≈600 and ≈100 kDa). PP1bps and PP1Cγ1 were enriched in the ≈600‐kDa peak, but PP1Cβ was enriched in the ≈100‐kDa peak. Furthermore, PP1bp175 and PP1bp134 exhibited lower binding of recombinant DIG‐PP1Cβ than recombinant DIG‐PP1Cγ1 or DIG‐PP1Cα. Solubilized PP1bp175 and PP1bp134 interact with PP1C under native conditions, because they both (1) coeluted from size‐exclusion and ion‐exchange columns; (2) bound to microcystin‐LR‐Sepharose; and (3) coprecipitated using PP1C antibodies. Trypsinolysis of the ≈600‐kDa form of PP1 increased phosphorylase a phosphatase activity approximately fourfold, suggesting that interaction of PP1C with these PP1bps modulates its activity. Thus, brain PP1 activity is likely targeted to the cytoskeleton, including postsynaptic densities, by isoform‐selective binding of PP1C to these targeting/regulatory subunits, contributing to the specificity of its physiological roles.
β‐Amyloid‐Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β‐Amyloid Precursor Protein
Authors: Le Weidong , Xie Wen Jie , Kong Rong , Appel Stanley H.
Published: July 2010

Abstract: Recent evidence suggests that β‐amyloid peptide (β‐AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β‐AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β‐AP1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF‐1, Tau‐1, and Tau‐5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF‐1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser199/202 and Ser396. In association with the increase in tau phosphorylation, the immunoreactivity of cell‐associated and secreted β‐amyloid precursor protein (β‐APP) was markedly elevated. Application of antisense oligonucleotide to β‐APP reduced expression of β‐APP and immunoreactivity of phosphorylated tau. Control peptide β‐AP1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell‐associated β‐APP. These results suggest that βAP1–40‐induced tau phosphorylation may be associated with increased β‐APP expression in degenerated neurons.
Increased Expression of Calbindin D28k via Herpes Simplex Virus Amplicon Vector Decreases Calcium Ion Mobilization and Enhances Neuronal Survival After Hypoglycemic Challenge
Authors: Meier Timothy J. , Ho Dora Y. , Sapolsky Robert M.
Published: July 2010

Abstract: Disruption of Ca2+ homeostasis often leads to neuron death. Recently, the function of calcium‐binding proteins as neuronal Ca2+ buffers has been debated. We tested whether calbindin D28k functions as an intracellular Ca2+ buffer by constructing bicistronic herpes simplex virus vectors to deliver rat calbindin cDNA to hippocampal neurons in vitro. Neurons were infected with vectors delivering calbindin or a negative control or were mock‐infected. After 12 or 24 h of hypoglycemia, infected cells were made aglycemic during fura‐2 calcium ratiometric imaging. In response to this challenge, neuronal overexpressing calbindin had less Ca2+ mobilized as compared with negative controls or mock‐infected cells. Cells were assayed for survival after 12‐ or 24‐h hypoglycemia or aglycemia. The calbindin vector decreased neuronal death due to hypoglycemia but not aglycemia. Here we demonstrate, in response to hypoglycemic challenge, both decreased Ca2+ mobilization and increased survival of cells infected with the calbindin vector.
Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B
Authors: Takagi Norio , Shinno Kiyohito , Teves Lucy , Bissoon Nankie , Wallace M. Christopher , Gurd James W.
Published: July 2010

Abstract: Activation of the N‐methyl‐d‐aspartate (NMDA) receptor has been implicated in the events leading to ischemia‐induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four‐vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti‐tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit‐specific antibodies followed by immunoblotting with anti‐phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29‐fold relative to sham‐operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia‐induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.
Lysophosphatidic Acid‐Induced Proliferation‐Related Signals in Astrocytes
Authors: Keller Jeffrey N. , Steiner Marion R. , Holtsberg Frederick W. , Mattson Mark P. , Steiner Sheldon M.
Published: July 2010

Abstract: Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA‐induced events in astrocytes were defined. As little as 1 nM LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca2+]i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca2+]i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca2+]i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca2+]i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA‐induced increase in [Ca2+]i was inhibited by a protein kinase C inhibitor, Ro 31‐8220, and a calcium‐dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca2+]i was important for the LPA‐induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA‐stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA‐induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin‐sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca2+]i, lipid peroxidation, and DNA synthesis.
Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum
Authors: Cadogan Anna‐Karina , Alexander Stephen P. H. , Boyd E. Andrew , Kendall David A.
Published: July 2010

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β(R)5α]‐(−)‐5‐(1,1‐dimethylheptyl)‐2‐[5‐hydroxy‐2‐(3‐hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [N‐(piperidin‐1‐yl)‐5‐(4‐chlorophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [3H]dopamine‐prelabelled striatal slices were also investigated. CP 55940 (1 µM) and anandamide (10 µM) caused significant reductions in forskolin‐stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µM). CP 55940 (1 µM) had no effect on either KCl‐ or neurotransmitter‐stimulated 3H‐inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [3H]dopamine‐prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.
Stimulation of Interleukin‐6 Secretion and Gene Transcription in Primary Astrocytes by Adenosine
Authors: Schwaninger Markus , Neher Mike , Viegas Elizabeth , Schneider Armin , Spranger Matthias
Published: July 2010

Abstract: During cerebral ischemia, the expression of interleukin‐6 (IL‐6), which has neuroprotective properties, increases. To understand the underlying mechanism, the regulation of IL‐6 expression by neurotransmitters that accumulate during cerebral ischemia was investigated. Adenosine stimulated IL‐6 secretion in primary astrocytes four‐ to 10‐fold. The effect was concentration dependent, the EC50 being ∼8 µM. Although the nonselective analogue 2‐chloroadenosine (2CA) increased IL‐6 secretion to a similar extent, the A1‐selective agonist N6‐cyclopentyladenosine or the A2a agonist CGS‐21680 had only a marginal effect on IL‐6 secretion. IL‐6 secretion stimulated by 2CA (10 µM) was inhibited by the nonselective adenosine antagonist 8‐(p‐sulfophenyl)theophylline, whereas the A1‐selective antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine or the A2a‐selective antagonist 8‐(3‐chlorostyryl)caffeine had no effect, to a concentration of 0.1 µM. Transcription of the IL‐6 gene was investigated by transfecting primary astrocytes with a reporter fusion gene containing the human IL‐6 promoter (−179/+12). 2CA stimulated IL‐6 gene transcription 2.5‐fold. Mutations of the binding site for NF‐κB or NF‐IL6 abrogated the response to 2CA. Thus, an increase of extracellular adenosine during focal cerebral ischemia may stimulate IL‐6 expression via A2b receptors. The induction of IL‐6 expression appears to involve a transcriptional effect that depends on NF‐κB and NF‐IL6.