bluuebunny/crossref_metadata_2025 huggingface dataset:
Corticosterone Has a Permissive Effect on Expression of Heme Oxygenase‐1 in CA1–CA3 Neurons of Hippocampus in Thermal‐Stressed Rats
Authors: Maines Mahin D. , Eke Benay C. , Weber Colleen M. , Ewing James F.
Published: July 2010

Abstract: Activity of the stress protein, heme oxygenase‐1 (hsp32; HO‐1), produces carbon monoxide (CO), the potential messenger molecule for excitatory N‐methyl‐d‐aspartate receptor‐mediated events, in the hippocampus. Long‐term stress caused by elevated adrenocorticoids induces pathological changes in CA1–CA3 neurons, of the hippocampus; the adrenal hormones also exacerbate damage from stress. In rats chronically treated with corticosterone, we examined expression of HO‐1 and its response to thermal stress in the hippocampus. An unprecedented appearance of scattered immunoreactive astrocytes marked the molecular layer of the hippocampus in corticosterone‐treated rats. Steroid treatment showed no discernible effect on whole‐brain HO‐1 mRNA. When these rats were subjected to hyperthermia, neurons in the CA1–CA3 area, including pyramidal cells, exhibited intense immunoreactivity for the oxygenase and a pronounced increase (∼10‐fold) in number. HO‐1 is essentially undetectable in this area when rats are exposed to chronic corticosterone alone or thermal stress by itself, or in control rats. In contrast, similar analysis of hilar neurons showed no apparent effect on either the number or relative intensity of HO‐1‐immunostained cells after treatment. Corticosterone treatment also intensified the stress response of cerebellum, including Purkinje cells and Bergmann glia in the molecular layer. In brain, despite a pronounced reduction in NO synthase activity in corticosterone‐treated and/or heat‐stressed animals, the level of cyclic GMP was not significantly reduced. These observations are consistent with the hypothesis that responsiveness to environmental stress of CA1–CA3 neurons brought about by chronic elevation in circulating adrenocorticoids results in an increased excitatory neuronal activity and eventual hippocampal degeneration. Moreover, these findings yield further support for a role of CO in the production of cyclic GMP in the brain.
In Human Fetal Astrocytes Exposure to Interleukin‐1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division
Authors: Lee Sunhee C. , Liu Wei , Dickson Dennis W. , Brosnan Celia F.
Published: July 2010

Abstract: In human astrocyte cultures established from second‐trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3+/glial fibrillary acidic protein (GFAP)+. The GD3+ cells were always GFAP+ and grew as flat, highly spread cells but changed to process‐bearing cells after interleukin‐1β (IL‐1β) stimulation. It is interesting that IL‐1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [3H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3+ population, however, consistently increased in absolute number after IL‐1β stimulation, in a dose‐ and time‐dependent manner. The IL‐1β‐mediated increase in number of GD3+ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL‐1β enhanced both the mean fluorescence intensity and the percentage of GD3+ cells. To investigate whether the increase in GD3+ astrocyte cell number was due to proliferation of preexisting GD3+ astrocytes or due to conversion of GD3− to GD3+ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double‐positive cells were extremely rare in both control and IL‐1β‐stimulated cultures. Moreover, an increase in number of GD3+ astrocytes was still observed in control and IL‐1β‐stimulated cultures where GD3+ cells had been initially eliminated by cell sorting. These results indicate that GD3+ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.
Arachidonic Acid, but Not Sodium Nitroprusside, Stimulates Presynaptic Protein Kinase C and Phosphorylation of GAP‐43 in Rat Hippocampal Slices and Synaptosomes
Authors: Luo Yuan , Vallano Mary Lou
Published: July 2010

Abstract: Activation of protein kinase C (PKC) and phosphorylation of its presynaptic substrate, the 43‐kDa growth‐associated protein GAP‐43, may contribute to the maintenance of hippocampal long‐term potentiation (LTP) by enhancing the probability of neurotransmitter release and/or modifying synaptic morphology. Induction of LTP in rat hippocampal slices by high‐frequency stimulation of Schaffer collateral‐CA1 synapses significantly increased the PKC‐dependent phosphorylation of GAP‐43, as assessed by quantitative immunoblotting with a monoclonal antibody that recognizes an epitope that is specifically phosphorylated by PKC. The stimulatory effect of high‐frequency stimulation on levels of immunoreactive phosphorylated GAP‐43 was not observed when 4‐amino‐5‐phosphonovalerate (50 µM), an N‐methyl‐d‐aspartate (NMDA) receptor antagonist, was bath‐applied during the high‐frequency stimulus. This observation supports the hypothesis that a retrograde messenger is produced postsynaptically following NMDA receptor activation and diffuses to the presynaptic terminal to activate PKC. Two retrograde messenger candidates—arachidonic acid and nitric oxide (sodium nitroprusside was used to generate nitric oxide)—were examined for their effects in hippocampal slices on PKC redistribution from cytosol to membrane as an indirect measure of enzyme activation and PKC‐specific GAP‐43 phosphorylation. Bath application of arachidonic acid, but not sodium nitroprusside, at concentrations that produce synaptic potentiation (100 µM and 1 mM, respectively) significantly increased translocation of PKC immunoreactivity from cytosol to membrane as well as levels of immunoreactive, phosphorylated GAP‐43. The stimulatory effect of arachidonic acid on GAP‐43 phosphorylation was also observed in hippocampal synaptosomes. These results indicate that arachidonic acid may contribute to LTP maintenance by activation of presynaptic PKC and phosphorylation of GAP‐43 substrate. The data also suggest that nitric oxide does not activate this signal transduction system and, by inference, activates a distinct biochemical pathway.
Evidence for Nerve Growth Factor‐Potentiating Activities of the Nonpeptidic Compound SR 57746A in PC12 Cells
Authors: Pradines Anne , Magazin Marilyn , Schiltz Pascal , Le Fur Gérard , Caput Daniel , Ferrara Pascual
Published: July 2010

Abstract: SR 57746A {1‐[2‐(naphth‐2‐yl)ethyl]‐4‐(3‐trifluoromethylphenyl)‐1,2,5,6‐tetrahydropyridine hydrochloride} exhibits neurotrophic activities in vivo and in vitro. We used the rat pheochromocytoma PC12 cell line to investigate in vitro cellular changes induced by SR 57746A. A significant increase in the percentage of cells bearing neurite‐like processes was obtained in cells treated by SR 57746A and nerve growth factor (NGF) compared with NGF treatment alone. SR 57746A added alone, however, had no effect on morphogenesis or on survival of cells in serum‐free medium. In contrast, SR 57746A induced a “priming” effect on PC12 cells for neurite outgrowth within 6 h of addition of the protein tyrosine kinase inhibitor genistein. An increase in α‐actinin content resulted from treatment with SR 57746A. Expression of NGF‐mediated acetylcholinesterase and choline acetyltransferase was enhanced within 5 days by SR 57746A. The molecule also induced rapid F‐actin redistribution. Within 2 min of incubation, outgrowth of F‐actin‐containing filopodia was clearly visible at the cell periphery, as previously shown with NGF. It is interesting that this effect of SR 57746A could be mimicked by protein tyrosine kinase inhibitors and abolished by preincubation with sodium orthovanadate, a protein tyrosine phosphatase inhibitor.
Fibroblasts that Reside in Mouse and Frog Injured Peripheral Nerves Produce Apolipoproteins
Authors: Saada A. , Dunaevsky‐Hutt A. , Aamar A. , Reichert F. , Rotshenker S.
Published: July 2010

Abstract: Apolipoprotein synthesis and secretion is upregulated in wallerian degenerating peripheral nerves. A commonly expressed view has been that macrophages are solely responsible for their production. In the present study we provide evidence that (1) nerve‐derived fibroblasts contribute to apolipoprotein production, (2) apolipoprotein production is confined to regions where myelin destruction and phagocytosis occur, and (3) some experimental procedures are detrimental for the production of apolipoproteins. Apolipoprotein production was studied in C57BL/6/NHSD (N) and C57/BL/6‐WLD/OLA/NHSD (W) mice that display, respectively, rapid and slow progression of wallerian degeneration. In N nerves, apolipoprotein E (apo‐E) is produced during in vitro and in vivo degeneration, and in vivo after freeze damage. In W nerves, apo‐E is produced at the injury region where degeneration occurs but not farther distally where degeneration fails to develop. Apo‐E is also produced in W nerves during in vitro degeneration and in vivo after freeze damage. In culture, N and W mice nerve‐derived fibroblasts, but neither macrophages nor Schwann cells produced apo‐E. Two apolipoproteins are produced in in vivo wallerian degenerating and freeze‐damaged frog nerves, i.e., apo‐39 and apo‐29. Only apo‐39 is produced in in vitro degenerating nerves. Neither apo‐39 nor apo‐29 is produced during in vivo degeneration in diffusion chambers. In culture, apo‐39 is produced by nerve‐derived fibroblasts and macrophages but not by Schwann cells.
NMDA Receptor Subtype Selectivity: Eliprodil, Polyamine Spider Toxins, Dextromethorphan, and Desipramine Selectively Block NMDA‐Evoked Striatal Acetylcholine but Not Spermidine Release
Authors: Nankai M. , Fage D. , Carter C.
Published: July 2010

Abstract: NMDA receptor stimulation concomitantly increases the release of [14C]acetylcholine and [3H]spermidine from rat striatal slices in vitro. The NMDA‐induced release of both acetylcholine and spermidine was blocked with equal potency by the NMDA channel blocker phencyclidine (0.1–10 µM). However, certain other channel blockers, including dextromethorphan (1–100 µM), which antagonized NMDA‐evoked acetylcholine release without affecting NMDA‐evoked spermidine release, and dextrorphan (1–100 µM) and memantine (1–100 µM), which block NMDA‐evoked acetylcholine release more potently than NMDA‐evoked spermidine release, showed greater selectivity of action. As previously shown for ifenprodil, eliprodil (SL82.0715; 1–100 µM) blocked NMDA‐evoked acetylcholine but not spermidine release. This selectivity is also observed for other agents interacting with the polyamine site(s) on the NMDA receptor, including arcaine (1–1,000 µM), philanthotoxin343, and argiotoxin636 (10 µM) and was also noted for desipramine (1–100 µM). The NMDA‐induced release of acetylcholine and spermidine is likely to be mediated by different native NMDA receptor subtypes, and several NMDA antagonists may be candidates for a selective action at a particular NMDA receptor subtype.
Cyclothiazide Modulates AMPA Receptor‐Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain
Authors: Hoyt Kari R. , Rajdev Sunita , Fattman Cheryl L. , Reynolds Ian J.
Published: July 2010

Abstract: We investigated the modulation of (±)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐induced increases in intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA‐induced changes in [Ca2+]i were increased by 0.3–100 µM cyclothiazide, with an EC50 value of 2.40 µM and a maximum potentiation of 428% of control values. [Ca2+]i responses to glutamate in the presence of N‐methyl‐d‐aspartate (NMDA) receptor antagonists were also potentiated by 10 µM cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non‐NMDA receptors. Almost all neurons responded with an increase in [Ca2+]i to both kainate and AMPA in the absence of extracellular Na+, and these Na+‐free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC50 value of 12.0 µM. Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA‐ and glutamate‐induced increases in [Mg2+]i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non‐NMDA glutamate receptors and may provide the basis for the increase in non‐NMDA receptor‐mediated excitotoxicity produced by cyclothiazide.
Protein Kinase C Modulates Calcium Channels in Isolated Presynaptic Nerve Terminals of Rat Hippocampus
Authors: Bartschat D. K. , Rhodes Troy E.
Published: July 2010

Abstract: Nerve terminals (“synaptosomes”) isolated from rat brain hippocampus were loaded with the fluorescent Ca2+ indicator fura‐2 and were subjected to depolarization with an elevated K+ concentration in a stopped‐flow spectrophotometer to measure the activity of voltage‐gated Ca2+ channels in the presynaptic membrane. Three components of Ca2+ influx were seen, which were tentatively identified as two classes of voltage‐dependent Ca2+ channels with different inactivation kinetics (τ of ∼60 ms and 1 s, respectively) and Na+/Ca2+ exchange working in the “reverse” mode. The activity of both classes of voltage‐dependent Ca2+ channels was slightly augmented by the phorbol ester phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage‐dependent Ca2+ channel activity, but the effect of DHS could be showed by coaddition of OKA. These results suggest that the activity of presynaptic voltage‐dependent Ca2+ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.
NMDA Receptor Blockade Increases In Vivo Striatal Dopamine Synthesis and Release in Rats and Mice with Incomplete, Dopamine‐Depleting, Nigrostriatal Lesions
Authors: Richard Matthew G. , Bennett James P.
Published: July 2010

Abstract: The role of excitatory amino acids (EAAs) in the regulation of striatal dopamine (DA) synthesis and release in rats and mice with incomplete DA‐depleting lesions of the nigrostriatal system was investigated using in vivo microdialysis in rats and estimates of striatal in vivo tyrosine hydroxylase activity in mice. Dopaminergic nigrostriatal input to the striatum was partially lesioned in rats with stereotactic perinigral 6‐hydroxydopamine injections and in C57BL mice with systemic 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) treatment. In rats, addition of the NMDA receptor antagonist 2‐amino‐5‐phosphonopentanoic acid (AP‐5, 10 mM) to dialysate increased local striatal DA release and synthesis. In 6‐hydroxydopamine partially lesioned rats, the effects of AP‐5 on DA release were significantly diminished, and AP‐5 had no significant effect on DA synthesis. Perfusion with the non‐NMDA receptor antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX, 1 mM) alone increased DA synthesis slightly, whereas DNQX + AP‐5 (10 mM) increased DA synthesis to levels comparable with those observed with AP‐5 alone. Local striatal DA synthesis was also increased by addition of the NMDA receptor antagonist (±)‐3‐(2‐carboxypiperizin‐4‐yl)propyl‐1‐phosphonic acid (CPP, 1 mM) to the perfusion buffer. Local inhibition of nitric oxide synthase with nitro‐l‐arginine (10 mM) perfused through the striatal microdialysis probe did not alter DA synthesis, suggesting that the effects observed with NMDA receptor blockade are not mediated by nitric oxide. In unlesioned mice, none of the systemically injected EAA receptor antagonists altered striatal DA synthesis. In MPTP‐treated mice with partial nigrostriatal lesions, treatment with the competitive NMDA receptor antagonist CPP (5 mg/kg i.p.) increased DA synthesis significantly compared with lesioned mice injected with vehicle. Our results suggest that EAAs mediate a tonic inhibition of DA terminal activity in the striatum via NMDA receptors and that this suppressive signal is reduced in animals with partial destruction of the nigrostriatal pathway.
Pituitary Adenylate Cyclase‐Activating Polypeptide: Control of Rat Pineal Cyclic AMP and Melatonin but Not Cyclic GMP
Authors: Chik Constance L. , Ho Anthony K.
Published: July 2010

Abstract: In this study, the effects of pituitary adenylate cyclase‐activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10−7M) increased MT production 2.5‐fold. PACAP (10−7M) also increased cyclic AMP accumulation four‐ to fivefold; this effect was potentiated two‐ to three‐fold by α1‐adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because α1‐adrenergic activation is known to translocate PKC and the PACAP‐stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4β‐phorbol 12‐myristate 13‐acetate (PMA). Phenylephrine and PMA also potentiated the PACAP‐stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an α1‐adrenergic → PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.
Transport of Gabapentin, a γ‐Amino Acid Drug, by System L α‐Amino Acid Transporters: A Comparative Study in Astrocytes, Synaptosomes, and CHO Cells
Authors: Su Ti‐Zhi , Lunney Elizabeth , Campbell Greg , Oxender Dale L.
Published: July 2010

Abstract: The system L transporter is generally considered to be one of the major Na+‐independent carriers for large neutral α‐amino acids in mammalian cells. However, we found that cultured astrocytes from rat brain cortex accumulate gabapentin, a γ‐amino acid, predominantly by this α‐amino acid transport system. Uptake of gabapentin by system L transporter was also examined in synaptosomes and Chinese hamster ovary (CHO) cells. The inhibition pattern displayed by various amino acids on gabapentin uptake in astrocytes and synaptosomes corresponds closely to that observed for the system L transport activity in CHO cells. Gabapentin and leucine have Km values that equal their Ki values for inhibition of each other, suggesting that leucine and gabapentin compete for the same system L transporter. By contrast, gabapentin exhibited no effect on uptake of GABA, glutamate, and arginine, indicating that these latter three types of brain transporters do not serve for uptake of gabapentin. A comparison of computer modeling analysis of gabapentin and l‐leucine structures shows that although the former is a γ‐amino acid, it can assume a conformation that can resemble the L‐form of a large neutral α‐amino acid such as l‐leucine. The steady‐state kinetic study in astrocytes and CHO cells indicates that the intracellular concentrations of gabapentin are about two to four times higher than that of leucine. The uptake levels of these two substrates are inversely related to their relative exodus rates. The concentrating ability by system L observed in astrocytes is consistent with the substantially high accumulation gradient of gabapentin in the brain tissue as determined by microdialysis.
Regionally Selective and Age‐Dependent Alterations in Benzodiazepine Receptor Binding in the Genetically Dystonic Hamster
Authors: Pratt Gerard D. , Richter Angelika , Möhler Hanns , Löscher Wolfgang
Published: July 2010

Abstract: Previous pharmacological studies have indicated that impairment of GABAergic transmission may be involved in the pathophysiology of dystonia in the mutant dtsz hamster, i.e., a genetic animal model for idiopathic dystonia. In the present experiments, the kinetic constants of [3H]flumazenil binding to the benzodiazepine site of the GABAA receptor were calculated from equilibrium binding measurements in various brain regions of genetically dystonic hamsters and age‐matched controls. Because dystonia in mutant dtsz hamsters is transient and disappears after ∼60–70 days of age, [3H]flumazenil binding was studied at the age of maximum severity of dystonia (30–40 days) and after disappearance of the disease, to examine which neurochemical changes were related to dystonia. In mutant hamsters with the maximum severity of dystonia, receptor affinity of [3H]flumazenil was increased in olfactory bulb,striatum, tectum, and cerebellum, as exemplified by significantly decreased dissociation constants (KD) in these regions. An increased number of binding sites (Bmax) were seen in striatum and frontal cortex but not in the other eight regions studied in this regard. All these changes in [3H]flumazenil binding disappeared in parallel with dystonia, implicating a causal relationship between altered benzodiazepine receptor binding and dystonia in mutant dtsz hamsters. In view of the antidystonic effect of benzodiazepines, such as diazepam, and recent neurochemical findings indicating impaired function of the GABA‐gated Cl− channel in dystonic hamsters, the present data might be interpreted as up‐regulation of benzodiazepine receptors in response to impaired GABAergic function. Furthermore, the present data represent the first evidence that GABAA receptors are altered in the basal ganglia in idiopathic (primary)dystonia.
Tellurite Specifically Affects Squalene Epoxidase: Investigations Examining the Mechanism of Tellurium‐Induced Neuropathy
Authors: Wagner Maria , Toews Arrel D. , Morell Pierre
Published: July 2010

Abstract: A peripheral neuropathy characterized by a transient demyelinating/remyelinating sequence results when young rats are fed a tellurium‐containing diet. The neuropathy occurs secondary to a systemic block in cholesterol synthesis. Squalene accumulation suggested the lesion was at the level of squalene epoxidase, a microsomal monooxygenase that uses NADPH cytochrome P450 reductase to receive its necessary reducing equivalents from NADPH. We have now demonstrated directly specificity for squalene epoxidase; our in vitro studies show that squalene epoxidase is inhibited 50% in the presence of 5 µM tellurite, the presumptive in vivo active metabolite. Under these conditions, the activities of other monooxygenases, aniline hydroxylase and benzo(a)pyrene hydroxylase, were inhibited less than 5%. We also present data suggesting that tellurite inhibits squalene epoxidation by interacting with highly susceptible ‐SH groups present on this monooxygenase. In vivo studies of specificity were based on the compensatory response to feeding of tellurium. Following tellurium intoxication, there was up‐regulation of squalene epoxidase activity both in liver (11‐fold) and sciatic nerve (fivefold). This induction was a specific response, as demonstrated in liver by the lack of up‐regulation following exposure to the nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also measured the microsomal monooxygenase activities of aniline hydroxylase and benzo(a)pyrene hydroxylase. Although they were induced following phenobarbital exposure, activities of these monooxygenases were not affected following tellurium intoxication, providing further evidence of specificity of tellurium intoxication for squalene epoxidase.
Variant Forms of Neuronal Glutamate Transporter Sites in Alzheimer's Disease Cerebral Cortex
Authors: Scott Heather L. , Tannenberg Anthony E. G. , Dodd Peter R.
Published: July 2010

Abstract: The displacement of Na+‐dependent d‐[3H]‐aspartate binding by unlabeled d‐aspartate or the inhibitors dl‐threo‐β‐hydroxyaspartate, l‐cysteate, l‐glutamate, dihydrokainate, dl‐α‐aminoadipate, α‐methyl‐dl‐glutamate, and 1‐aminocyclobutane‐cis‐1,3‐dicarboxylate was used to characterize the high‐affinity glutamate/aspartate uptake site in human cerebral cortex. Synaptosomal membranes were prepared from tissue obtained at autopsy from nondemented control, Alzheimer's disease (AD), and diffuse Lewy body disease (DLBD) cases. Areas that are damaged in AD (midtemporal, frontal, caudal cingulate, and hippocampal cortices) were compared with those that are spared (occipital and motor cortices). Profiles of the affinities (Ka values) of the ligands showed that areas spared from damage in AD cases differed significantly from equivalent areas in control (p < 0.001) and DLBD (p < 0.001) cases and also from areas susceptible to damage in the same AD cases (p < 0.001). Areas susceptible to damage in AD showed comparable profiles across the three case groups (p = 0.980). The glutamate/aspartate uptake site may be regionally variant in AD cases, and this may underlie local excitotoxicity. d‐[3H]Aspartate binding site density was significantly lower in both dementia groups (control vs. AD, p < 0.001; control vs. DLBD, p = 0.009; but AD vs. DLBD, p = 0.528); within‐group differences were not significant (control, p = 0.874; AD, p = 0.285; DLBD, p = 0.741).
Liposome‐Mediated Gene Transfer into Normal and Dystrophin‐Deficient Mouse Myoblasts
Authors: Trivedi Rikin A. , Dickson George
Published: July 2010

Abstract: A range of tissue types has now been targeted for development of gene therapeutic procedures both to correct genetic defects and to treat acquired disease. In particular, skeletal muscle holds great importance, not exclusively for the treatment of inherited muscle disorders but also as a platform for the expression of heterologous recombinant proteins, destined to immunise the host or to serve some systemic therapeutic goal. With respect to the X‐linked myopathy Duchenne muscular dystrophy (DMD), several gene therapy protocols are being developed that focus on complementing primary genetic defects in the DMD gene by introducing copies of recombinant gene constructs into muscle cells both ex vivo and in vivo. In the present study the potential use of a range of polycationic liposomes as physical gene delivery systems for skeletal muscle has been examined. Using a LacZ reporter gene under optimised conditions up to 40% transfection efficiencies were obtained with the mouse myoblast cell line C2C12. With primary cultures of normal and dystrophin‐deficient mdx mouse muscle, up to 10% transfection efficiency was obtained with reporter gene constructs, and high levels of recombinant human dystrophin expression were observed following transfer of dystrophin cDNA gene constructs. These in vitro studies indicate that cationic liposomes can be used to deliver recombinant genes to muscle cells at high efficiency and form a basis to expand investigations into in vivo expression of recombinant dystrophin protein either by direct intramuscular gene transfer or via implantation of transfected myoblasts.
Involvement of Free Radicals in Excitotoxicity In Vivo
Authors: Schulz Jörg B. , Henshaw D. Ross , Siwek Donald , Jenkins Bruce G. , Ferrante Robert J. , Cipolloni P. Ben , Kowall Neil W. , Rosen Bruce R. , Beal M. Flint
Published: July 2010

Abstract: Recent evidence has linked excitotoxicity with the generation of free radicals. We examined whether free radical spin traps can attenuate excitotoxic lesions in vivo. Pretreatment with N‐tert‐butyl‐α‐(2‐sulfophenyl)‐nitrone (S‐PBN) significantly attenuated striatal excitotoxic lesions in rats produced by N‐methyl‐d‐aspartate (NMDA), kainic acid, and α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid (AMPA). In a similar manner, striatal lesions produced by 1‐methyl‐4‐phenylpyridinium (MPP+), malonate, and 3‐acetylpyridine were significantly attenuated by either S‐PBN or α‐phenyl‐N‐tert‐butylnitrone (PBN) treatment. Administration of S‐PBN in combination with the NMDA antagonist MK‐801 produced additive effects against malonate and 3‐acetylpyridine toxicity. Malonate injections resulted in increased production of hydroxyl free radicals (•OH) as assessed by the conversion of salicylate to 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA). This increase was significantly attenuated by S‐PBN, consistent with a free radical scavenging effect. S‐PBN had no effects on malonate‐induced ATP depletions and had no significant effect on spontaneous striatal electrophysiologic activity. These results provide the first direct in vivo evidence for the involvement of free radicals in excitotoxicity and suggest that antioxidants may be useful in treating neurologic illnesses in which excitotoxic mechanisms have been implicated.
The Effect of Chronic Treatment with the GABA Transaminase Inhibitors γ‐Vinyl‐GABA and Ethanolamine‐O‐Sulphate on the In Vivo Release of GABA from Rat Hippocampus
Authors: Qume M. , Whitton P. S. , Fowler L. J.
Published: July 2010

Abstract: The effects of chronic treatment with the specific, mechanism‐based, irreversible inhibitors of 4‐aminobutyrate aminotransferase (EC 2.6.1.19; GABA transaminase), ethanolamine O‐sulphate (EOS), and 4‐aminohexenoate [vigabatrin; γ‐vinyl‐GABA (GVG)] on the extracellular concentrations of GABA in the hippocampus have been studied using in vivo microdialysis in conscious animals. Oral dosing [3 mg/ml of drinking water, giving doses of GVG of 194 ± 38 mg/kg/day and of EOS of 303 ± 42 mg/kg/day (mean ± SD)] was followed by microdialysis at 2, 8, and 21 days. The basal outflow of GABA (in the range of ∼1–2 pmol/30 µl/30‐min sample) after 2 and 8 days of treatment was not significantly different from that in control animals, but the 21‐day treatment gave significant rises in the extracellular GABA concentration (up to ∼6–8 pmol/30 µl/30‐min sample). Both inhibitors gave similar results. Depolarisation with 100 mM K+ gave large increases in GABA release in control (∼20–60 pmol/30 µl/30‐min sample) and treated animals. The 8‐ and 21‐day‐treated animals showed significant increases in the stimulated release compared with control animals (∼80–100 pmol/30 µl/30‐min sample). Excluding Ca2+ had no significant effect on either basal or stimulated release. The significant increases in K+‐evoked release of GABA show that the increased intracellular pool of GABA is available for release, and this may be related to the anticonvulsant action of these compounds.
Interaction Between Adrenergic and Peptide Stimulation in the Rat Pineal: Pituitary Adenylate Cyclase‐Activating Peptide
Authors: Yuwiler A. , Brammer G. L. , Bennett B. L.
Published: July 2010

Abstract: The 27 amino acid peptide, pituitary adenylate cyclase‐activating polypeptide (PACAP‐27), and its 38 amino acid analogue, PACAP‐38, stimulate serotonin‐N‐acetyltransferase (NAT) activity and N‐acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP‐38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N‐terminal histidine C‐terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP‐38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP‐38 reduces the magnitude of subsequent stimulation with PACAP‐38 or forskolin. Concurrent stimulation of α‐receptors or treatment with active phorbol ester augments rat pineal response to PACAP‐38 stimulation just as it increases the response to VIP, PHI, and β‐receptor stimulation. Pineals from newborn rats respond to PACAP‐38 with an increase in NAT activity and the increase is augmented by concomitant α1‐adrenergic stimulation. The putative PACAP inhibitor PACAP (6–38) and the putative VIP inhibitor (Ac‐Tyr,d‐Phe)‐GRF 1–29 amide, in 100–1,000‐fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.
Regulation of Sialyltransferase Activities by Phosphorylation and Dephosphorylation
Authors: Gu Xinbin , Preuß Ute , Gu Tianjue , Yu Robert K.
Published: July 2010

Abstract: The composition of tissue gangliosides is thought to result mainly from the active regulation and selective expression of specific enzymes responsible for their metabolism. In the last few years, we have purified several rat brain sialyltransferases to homogeneity; the availability of these highly purified enzymes enabled us to investigate their regulation and expression at the molecular level. Thus, we studied the regulation of sialyltransferase activities, in particular, CMP‐NeuAc:GM1 and CMP‐NeuAc:LacCer sialyltransferases by a phosphorylation/dephosphorylation mechanism. Protein kinase C was added to a standard enzyme assay mixture containing [γ‐32P]ATP, and the activity of the enzyme was measured after various incubation times. We found that treatment of several sialyltransferases by protein kinase C decreased their activities in a time‐dependent manner. Analyses of 32P‐labeled amino acids revealed that the major phosphorylation site of CMP‐NeuAc:GM1 α2→3 sialyltransferase (ST‐IV) was serine and that for CMP‐NeuAc:LacCer α2→3 sialyltransferase (ST‐I) was primarily threonine. Partial recovery of the enzyme activity could be achieved by treatment of the phosphorylated sialyltransferases with rat brain protein phosphatase. We conclude that the activities of sialyltransferases can be modulated by protein kinase C and protein phosphatase and this may represent a potential regulatory mechanism for ganglioside biosynthesis.
Immunocytochemical Examination of Neural Rat and Mouse Primary Cultures Using Monoclonal Antibodies Raised Against Pyruvate Carboxylase
Authors: Cesar Marija , Hamprecht Bernd
Published: July 2010

Abstract: Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy‐dependent fixation of CO2 to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia‐rich and neuron‐rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia‐rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP‐positive cells showing no specific staining for PC, and vice versa. Also, in neuron‐rich primary cultures PC was found only in the ∼10% GFAP‐expressing astroglial cells contaminating the neuron‐rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron‐specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain.
Characterization of a Cloned Locust Tyramine Receptor cDNA by Functional Expression in Permanently Transformed Drosophila S2 Cells
Authors: Broeck J. Vanden , Vulsteke V. , Huybrechts R. , De Loof A.
Published: July 2010

Abstract: The cDNA for Tyr‐Loc, a G protein‐coupled receptor that clearly shows homology to a number of mammalian and fruit fly receptors for biogenic amines, was cloned from the nervous system of Locusta migratoria. Functional expression of the cloned cDNA was obtained in cultured insect cells, i.e., in Spodoptera SF9 cells using a baculoviral expression system and in stably transformed Drosophila Schneider 2 (S2) cells. Multiple copies of the receptor expression construct are inserted into the genome of these permanently transformed cells. The expression of the receptor cDNA was driven by the upstream sequences of a Bombyx mori baculoviral immediate early gene. Tyramine shows a much higher binding affinity to this receptor than other possible endogenous ligands. It also reduces forskolin‐induced cyclic AMP production in the permanently transformed S2 cells. The pharmacological profile of the Tyr‐Loc receptor is distinct from that of any locust receptor‐type described so far, but it is similar to that of the Drosophila tyramine/octopamine receptor. In the locust CNS, the Tyr‐Loc mRNA is not present in the distal part of the optic lobes but has a widespread distribution in the brain and the ventral nerve cord.
Regulation of Tyrosine Hydroxylase Gene Expression in IMR‐32 Neuroblastoma Cells by Basic Fibroblast Growth Factor and Ciliary Neurotrophic Factor
Authors: Rabinovsky Eric D. , Ramchatesingh Jackie , McManaman James L.
Published: July 2010

Abstract: The actions of basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) on tyrosine hydroxylase (TH) gene expression were studied using IMR‐32 neuroblastoma cells. Treatment of these cells with bFGF for 3 days induced the expression of detectable levels of immunoreactive TH protein and TH mRNA. In contrast, CNTF did not affect TH expression unless bFGF was present. In the presence of saturating amounts of bFGF, CNTF increased TH protein and mRNA levels of TH two‐ to threefold over those found in bFGF‐treated cultures. The effects of CNTF on TH expression diminished with increasing culture time, and after 6 days of incubation CNTF no longer enhanced TH levels. The requirement for bFGF as cofactor in the effects of CNTF on TH was specific, as CNTF did not affect TH when it was coadministered with 8‐(4‐chlorophenylthio)‐cyclic AMP, another agent that stimulates TH development in this cell line, and bFGF was not required for CNTF to stimulate the development of choline acetyltransferase. Moreover, cotreatment with bFGF reduced the ability of CNTF to enhance choline acetyltransferase. These results demonstrate that bFGF and CNTF can enhance expression of TH and that bFGF can modify the effects of CNTF on neurotransmitter phenotype.
Human Microtubule‐Associated Protein‐2c Localizes to Dendrites and Axons in Fetal Spinal Motor Neurons
Authors: Albala Joanna S. , Kress Yvonne , Liu Wan‐Kyng , Weidenheim Karen , Yen Shu‐Hui C. , Shafit‐Zagardo Bridget
Published: July 2010

Abstract: Microtubule‐associated protein‐2 (MAP‐2) functions to maintain neuronal morphology by promoting the assembly of microtubules. MAP‐2c is an alternately spliced form of MAP‐2, containing the first 151 amino acids of high‐molecular‐weight (HMW) MAP‐2 joined to the last 321 amino acids, eliminating 1,352 amino acids specific to HMW MAP‐2. A polyclonal antibody generated to the splice site of human MAP‐2c was used to determine its cellular localization. The MAP‐2c antiserum was depleted of any HMW MAP‐2 reactivity by absorption with HMW MAP‐2 fusion protein. Western blot analysis of human fetal spinal cord homogenates demonstrated that the antibody is specific for human MAP‐2c. MAP‐2c immunoreactivity was found in the perinuclear cytoplasm and processes of anterior motor neurons and large processes of the posterior column in sections from 22–24‐week human fetal spinal cord. Double‐label confocal microscopy was performed using the MAP‐2c polyclonal antibody and either a HMW MAP‐2 or a neurofilament protein (highly phosphorylated 160‐ and 200‐kDa protein) monoclonal antibody to identify these processes as dendrites or axons, respectively. HMW MAP‐2 and MAP‐2c colocalized in cell bodies and dendrites of anterior motor neurons, demonstrating for the first time the presence of native MAP‐2c within dendrites. In addition, immunoelectron microscopy showed MAP‐2c associated with microtubules in dendrites of motor neurons. MAP‐2c and the neurofilament proteins were found in axons of the dorsal and ventral roots. The presence of MAP‐2c within axons and dendrites suggests that MAP‐2c contributes to neuronal plasticity during human fetal development.
Pigment Epithelium‐Derived Factor Is a Survival Factor for Cerebellar Granule Cells in Culture
Authors: Taniwaki Takayuki , Becerra S. Patricia , Chader Gerald J. , Schwartz Joan P.
Published: July 2010

Abstract: Pigment epithelium‐derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y‐79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose‐response relationship, with a larger effect in chemically defined medium than in serum‐containing medium [ED50 = 30 ng/ml (0.70 nM) in chemically defined medium and 100 ng/ml (2.3 nM) in serum‐containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme‐linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.
Lack of Rapid Desensitization of Ca2+ Responses in Transfected CHO Cells Expressing the Rat Neurotensin Receptor Despite Agonist‐Induced Internalization
Authors: Hermans Emmanuel , Gailly Philippe , Gillis Jean Marie , Octave Jean‐Noël , Maloteaux Jean‐Marie
Published: July 2010

Abstract: Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca2+]i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca2+]i resulted from release of Ca2+ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca2+]i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for ∼70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells. A possible absence of desensitization of the neurotensin receptor itself is also discussed.
Opposite Regulation of Basic Fibroblast Growth Factor and Nerve Growth Factor Gene Expression in Rat Cortical Astrocytes Following Dexamethasone Treatment
Authors: Riva M. A. , Fumagalli F. , Racagni G.
Published: July 2010

Abstract: Growth factors are peptides that exert different activities in the CNS, supporting the survival of different cell populations and playing an important role in the maintenance of cell homeostasis. Much evidence has suggested that these molecules can protect neurons from degeneration induced by mechanical injury or excitotoxic stimuli. Different factors can contribute to the regulation of neurotrophic factor expression in the brain. Such mechanisms may therefore be important in the manipulation of the levels of these peptides in specific brain areas as a therapeutic intervention in acute and chronic neurodegenerative diseases. We have used a primary culture of rat cortical astrocytes to investigate the regulation of basic fibroblast growth factor (bFGF) gene expression in comparison with other neurotrophic molecules. Our results indicate that the glucocorticoid analogue dexamethasone markedly elevates bFGF mRNA levels but reduces the expression of nerve growth factor. The induction of bFGF was transient, as it peaked after 6 h and returned to basal levels within 24 h and was not blocked by coincubation of cycloheximide, thus indicating that it did not require de novo protein synthesis. This effect was also observed in vivo, as systemic injection of dexamethasone (1 or 10 mg/kg) produced a significant increase in the amount of bFGF mRNA in cerebral cortex and hippocampus. The effect we describe can contribute to the regulation of bFGF expression in the brain and may be important in relation to the protective effect exerted by this growth factor in different models of neuronal injury.
Expression of the μ‐Opioid Receptor in CHO Cells: Ability of μ‐Opioid Ligands to Promote α‐Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits
Authors: Chakrabarti Sumita , Prather Paul L. , Yu Lei , Law Ping‐Yee , Loh Horace H.
Published: July 2010

Abstract: The identities of heterotrimeric G proteins that can interact with the μ‐opioid receptor were investigated by α‐azidoanilido[32P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)‐MORIVA3 cells, a CHO clone that stably expressed μ‐opioid receptor cDNA (MOR‐1). This clone expressed 1.01 × 106μ‐opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ‐opioid‐selective ligands [d‐Ala2,N‐MePhe4,Gly‐ol]‐enkephalin and [N‐MePhe3,d‐Pro4]‐morphiceptin, relative to the δ‐selective opioid agonist [d‐Pen2,d‐Pen5]‐enkephalin or the κ‐selective opioid agonist U‐50,488H. μ‐Opioid ligands induced an increase in α‐azidoanilido[32P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as Gi3α, Gi2α, and Go2α. The same pattern of simultaneous interaction of the μ‐opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10‐fold fewer receptors as those expressed in CHO‐MORIVA3 cells. The opioid‐induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist‐induced increase of α‐azidoanilido[32P]GTP incorporation into Gi2α (160–280%) and Go2α (110–220%) than for an unknown Gα (G?α) (60%) or Gi3α (40%) was produced by three different μ‐opioid ligands tested. In addition, slight differences were also found between the ability of various μ‐opioid agonists to produce half‐maximal labeling (ED50) of any given Gα subunit, with a rank order of Gi3α > Go2α > Gi2α = G?α. In any case, these results suggest that the activated μ‐opioid receptor couples to four distinct G protein α subunits simultaneously.