bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of [3H]Quisqualate Binding to Recombinant Rat Metabotropic Glutamate 1a and 5a Receptors and to Rat and Human Brain Sections
Authors: Mutel Vincent , Ellis Gareth J. , Adam Geo , Chaboz Sylvie , Nilly Agnes , Messer Jürg , Bleuel Zaiga , Metzler Veit , Malherbe Pari , Schlaeger Ernst‐Jürgen , Roughley Brian S. , Faull Richard L. M. , Richards J. Grayson
Published: March 2003

Abstract: We have investigated the binding properties of[3H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5areceptors and to rat and human brain sections. Saturation isotherms gaveKD values of 27 ± 4 and 81 ± 22 nMfor mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited thebinding to mGlu1a and mGlu5a receptors concentration‐dependently.(S)‐4‐Carboxyphenylglycine,(S)‐4‐carboxy‐3‐hydroxyphenylglycine, and(R,S)‐1‐aminoindan‐1,5‐dicarboxylic acid, which completely inhibited[3H]quisqualate binding to the mGlu5a receptor, were inactive in afunctional assay using this receptor. The distribution and abundance ofbinding sites in rat and human brain sections were studied by quantitativereceptor radioautography and image analysis. Using 10 nM[3H]quisqualate, a high density of binding was detected in variousbrain regions with the following rank order of increasing levels: medulla,thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactorytubercle, dentate gyrus molecular layer, CA1‐3 oriens layer of hippocampus,striatum, and cerebellar molecular layer. The ionotropic component of thisbinding could be inhibited by 30 μM kainate, revealing thedistribution of mGlu1+5 receptors. The latter were almost completely inhibitedby the group I agonist (S)‐3,5‐dihydroxyphenylglycine. The bindingprofile correlated well with the cellular sites of synthesis and regionalexpression of the respective group I receptor proteins revealed by in situhybridization histochemistry and immunohistochemistry, respectively.
Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata
Authors: Furukawa Nobuya , Arai Nobutaka , Goshima Yoshio , Miyamae Takeaki , Ohshima Etsuo , Suzuki Fumio , Fujita Kiyohide , Misu Yoshimi
Published: March 2003

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four‐vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal l‐aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10–30 nm DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration‐dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nm, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration‐dependently by intrastriatal perfusion of 0.3–1 mm DOPA and stereoselectively by 0.6 mm DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.
Brain‐derived neurotrophic factor mediates an excitoprotective effect of dietary restriction in mice
Authors: Duan Wenzhen , Guo ZhiHong , Mattson Mark P.
Published: March 2003

Dietary restriction (DR; reduced calorie intake) increases the lifespan of rodents and increases their resistance to cancer, diabetes and other age‐related diseases. DR also exerts beneficial effects on the brain including enhanced learning and memory and increased resistance of neurons to excitotoxic, oxidative and metabolic insults. The mechanisms underlying the effects of DR on neuronal plasticity and survival are unknown. In the present study we show that levels of brain‐derived neurotrophic factor (BDNF) are significantly increased in the hippocampus, cerebral cortex and striatum of mice maintained on an alternate day feeding DR regimen compared to animals fed ad libitum. Damage to hippocampal neurons induced by the excitotoxin kainic acid was significantly reduced in mice maintained on DR, and this neuroprotective effect was attenuated by intraventricular administration of a BDNF‐blocking antibody. Our findings show that simply reducing food intake results in increased levels of BDNF in brain cells, and suggest that the resulting activation of BDNF signaling pathways plays a key role in the neuroprotective effect of DR. These results bolster accumulating evidence that DR may be an effective approach for increasing the resistance of the brain to damage and enhancing brain neuronal plasticity.
Gas chromatography–mass spectrometry analysis of endogenous cannabinoids in healthy and tumoral human brain and human cells in culture
Authors: Maccarrone Mauro , Attinà Marina , Cartoni Antonella , Bari Monica , Finazzi‐Agrò Alessandro
Published: March 2003

Endocannabinoids are lipid mediators thought to modulate central and peripheral neural functions. We report here gas chromatography–electron impact mass spectrometry analysis of human brain, showing that lipid extracts contain anandamide and 2‐arachidonoylglycerol (2‐AG), the most active endocannabinoids known to date. Human brain also contained the endocannabinoid‐like compounds N‐oleoylethanolamine, N‐palmitoylethanolamine and N‐stearoylethanolamine. Anandamide and 2‐AG (0.16 ± 0.05 and 0.10 ± 0.05 nmol/mg protein, respectively) represented 7.7% and 4.8% of total endocannabinoid‐like compounds, respectively. N‐Palmitoyethanolamine was the most abundant (50%), followed by N‐oleoyl (23.6%) and N‐stearoyl (13.9%) ethanolamines. A similar composition in endocannabinoid‐like compounds was found in human neuroblastoma CHP100 and lymphoma U937 cells, and also in rat brain. Remarkably, human meningioma specimens showed an approximately six‐fold smaller content of all N‐acylethanolamines, but not of 2‐AG, and a similar decrease was observed in a human glioblastoma. These ex vivo results fully support the purported roles of endocannabinoids in the nervous system.
α‐Synuclein forms a complex with transcription factor Elk‐1
Authors: Iwata Atsushi , Miura Seiko , Kanazawa Ichiro , Sawada Makoto , Nukina Nobuyuki
Published: March 2003

α‐Synuclein has been identified as a component of Lewy bodies in Parkinson's disease and diffuse Lewy body disease, and glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA). To explore the role of α‐synuclein in the pathogenesis, we searched for molecules interacting with α‐synuclein and discovered that GCIs are stained by anti‐Elk‐1 antibody. To seek the role of Elk‐1 in synucleinopathies, we cotransfected α‐synuclein and Elk‐1 to cultured cells, and found small granular structure complexes where the two molecules colocalized. Moreover, α‐synuclein and Elk‐1 were co‐immunoprecipitated from the cell lysates. For formation of the complex, the presence of both ETS and B‐box domains of Elk‐1 was required. Although there was no evidence of direct binding between α‐synuclein and Elk‐1, we discovered that α‐synuclein and Elk‐1 both bind to ERK‐2, a MAP kinase. The effect of α‐synuclein on the MAP kinase pathway was assessed using the Pathdetect system, which showed prominent attenuation of Elk‐1 phosphorylation with α‐synuclein, and especially A53T mutant. Our results suggest that α‐synuclein reacts with the MAP kinase pathway, which might cause dysfunction of neurons and oligodendrocytes and lead to neurodegeneration in Parkinson's disease and MSA.
Human immunodeficiency virus type 1 Tat protein decreases cyclic AMP synthesis in rat microglia cultures
Authors: Patrizio Mario , Colucci Mariantonella , Levi Giulio
Published: March 2003

We have studied the modulation of cyclic AMP (cAMP) accumulation by the human immunodeficiency virus type 1 (HIV 1) protein Tat in microglia and astrocyte cultures obtained from neonatal rat brain. Pretreatment of microglia with recombinant Tat resulted in a dose‐ and time‐dependent decrease of cAMP accumulation induced by subsequent exposure to isoproterenol (1 µm). The inhibitory action of 100 ng/mL Tat approached 50% after 4 h of preincubation and reached a maximum of 70% after 24 h. The Tat‐induced time‐ and dose‐dependent decrease of cAMP accumulation was observed also when microglial cultures were stimulated with the adenylyl cyclase activator forskolin (100 µm). In both cases, Tat inhibitory action was 70% reverted by a specific monoclonal anti‐Tat antibody, but was not prevented either by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methyl‐xantine (100 µm) or by a 16‐h pretreatment of microglial cultures with the Gi protein inhibitor pertussis toxin (10 ng/mL). All these results suggested that the viral protein acts at a step of the cAMP transduction pathway other than receptors, G proteins and phosphodiesterases. The target of Tat appeared to be adenylyl cyclase, whose activity was markedly reduced (up to 60%) in membranes prepared from Tat‐treated microglial cells, both in basal conditions and after stimulation with isoproterenol and forskolin. The inability of the competitive inhibitor of nitric oxide synthase NG‐monometyl‐

l‐arginine (20 and 200 µm) to revert Tat action on forskolin‐induced cAMP accumulation, and of two potent nitric oxide donors, PAPA and DETA (0.1–2 m

m), to alter forskolin‐induced cAMP accumulation, excluded an involvement of nitric oxide in Tat‐induced adenylyl cyclase inhibition. On the contrary, two inhibitors of nuclear factor κB activation, N‐tosyl‐l‐phenylalanine chloromethyl ketone (10 µm) and SN50 (25 µm), markedly prevented the reduction of forskolin‐evoked cAMP accumulation by Tat, suggesting a possible role for this nuclear transcriptional factor in the regulation of adenylyl cyclase by Tat in microglia. This assumption was strengthened by the ability of lipopolysaccharide (100 ng/mL, 4 h) to mimic the inhibitory effect of the viral protein. Conversely, astrocyte cAMP accumulation was unaffected by the viral protein, as tested at various concentrations and time points. Finally, Tat inhibition of microglial adenylyl cyclase was not due to non‐specific cytotoxicity. As cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, and microglia is believed to mediate Tat‐induced neurotoxicity, these results suggest that the ability of Tat to inhibit cAMP synthesis in microglia may contribute to neuronal degeneration and cell death associated with HIV infection.
Characterization of ‘non‐N‐methyl‐d‐Aspartate’ binding sites for gacyclidine enantiomers in the rat cerebellar and telencephalic structures
Authors: Hirbec Hélène , Kamenka Jean Marc , Privat Alain , Vignon Jacques
Published: March 2003

Gacyclidine is a non‐competitive NMDA receptor antagonist with potent neuroprotective properties. However, we have previously demonstrated that gacyclidine enantiomers [(–) and (+)GK11] interact with other (‘non‐NMDA’) binding sites which may play a role in the lower self‐neurotoxicity of this compound relative to the other NMDA receptor antagonists. Evidence for these binding sites has been obtained from autoradiographic and membrane binding experiments. They were found to be expressed at high levels in the molecular layer of the cerebellum, although they can also been seen in the granular layer and in telencephalic regions. The present study was designed to further characterize these gacyclidine ‘non‐NMDA’ binding sites. The pharmacological profiles obtained on cerebellar and telencephalic membrane homogenates showed that they could not be linked directly to the main receptors or uptake complexes of the central nervous system (CNS). However, the comparison of (–) and (+)[3H]GK11 binding distribution in different mutant animals bearing specific cellular deficits in the cerebellum has demonstrated that the gacyclidine ‘non‐NMDA’ binding sites are associated with the dendritic trees of Purkinje cells. Interestingly, our study also shows that the radioligand binding to both cerebellar and telencephalic structures could be modulated by endogeneous factors which can be removed by a stringent prewashing procedure.
Hypoleptinemia, but not hypoinsulinemia, induces hyperphagia in streptozotocin‐induced diabetic rats
Authors: Hidaka Shuji , Yoshimatsu Hironobu , Kondou Seiya , Oka Kyoko , Tsuruta Yoshio , Sakino Hiroshi , Itateyama Emi , Noguchi Hitoshi , Himeno Katsurou , Okamoto Kenjirou , Teshima Yasushi , Okeda Toshimitsu , Sakata Toshiie
Published: March 2003

To assess the dominance between hypoinsulinemia and hypoleptinemia as factors in the development of hyperphagia in streptozotocin (STZ)‐induced diabetes mellitus (STZ‐DM) rodents with respect to hormone–neuropeptide interactions, changes in gene expression of agouti gene‐related protein (AGRP) in the arcuate nucleus of the hypothalamus were investigated using STZ‐DM rats, fasting Zucker fa/fa rats and STZ‐DM agouti (STZ‐DM Ay/a) mice. AGRP mRNA and neuropeptide Y mRNA were both significantly up‐regulated in STZ‐DM rats, which are associated with body weight loss, hyperglycemia, hypoinsulinemia and hypoleptinemia. We proceeded to analyze whether insulin or leptin played the greater role in the regulation of AGRP using Zucker fa/fa rats. The AGRP mRNA did not differ significantly between fasted fa/fa rats, which have both leptin‐insensitivity and hypoinsulinemia, and fed Zuckers, which have leptin‐insensitivity and hyperinsulinemia. We further found that up‐regulation of AGRP expression was normalized by infusion of leptin into the third cerebroventricle (i3vt), but not by i3vt infusion of insulin, although up‐regulation of AGRP was partially corrected by systemic insulin infusion. The latter finding supports hypoleptinemia as a key‐modulator of STZ‐DM‐induced hyperphagia because systemic insulin infusion, at least partially, restored hypoleptinemia through its acceleration of fat deposition, as demonstrated by the partial recovery of lost body weight. After STZ‐DM induction, Ay/a mice whose melanocortin‐4 receptor (MC4‐R) was blocked by ectopic expression of agouti protein additionally accelerated hyperphagia and up‐regulated AGRP mRNA, implying that the mechanism is triggered by a leptin deficit rather than by the main action of the message through MC4‐R. Hypoleptinemia, but not hypoinsulinemia per se, thus develops hyperphagia in STZ‐DM rodents. These results are very much in line with evidence that hypothalamic neuropeptides are potently regulated by leptin as downstream targets of its actions.
C‐terminal fragment of amyloid precursor protein induces astrocytosis
Authors: Bach Jae‐Hyung , Chae Hee Sun , Rah Jong Choel , Lee Myoung Woo , Park Cheol Hyoung , Choi Se Hoon , Choi Jeong Kyu , Lee Sang Hyung , Kim Yong Sik , Kim Kyung Yong , Lee Won Bok , Suh Yoo‐Hun , Kim Sung Su
Published: March 2003

One of the pathophysiological features of Alzheimer's disease is astrocytosis around senile plaques. Reactive astrocytes may produce proinflammatory mediators, nitric oxide, and subsequent reactive oxygen intermediates such as peroxynitrites. In the present study, we investigated the possible role of the C‐terminal fragment of amyloid precursor protein (CT‐APP), which is another constituent of amyloid senile plaque and an abnormal product of APP metabolism, as an inducer of astrocytosis. We report that 100 nm recombinant C‐terminal 105 amino acid fragment (CT105) of APP induced astrocytosis morphologically and immunologically. CT105 exposure resulted in activation of mitogen‐activated protein kinase (MAPK) pathways as well as transcription factor NF‐κB. Pretreatment with PD098059 and/or SB203580 decreased nitric oxide (NO) production and nuclear factor‐kappa B (NF‐κB) activation. But inhibitors of NF‐κB activation did not affect MAPKs activation whereas they abolished NO production and attenuated astrocytosis. Furthermore, conditioned media derived from CT105‐treated astrocytes enhanced neurotoxicity and pretreatment with NO and peroxynitrite scavengers attenuated its toxicity. These suggest that CT‐APP may participate in Alzheimer's pathogenesis through MAPKs‐ and NF‐κB‐dependent astrocytosis and iNOS induction.
Coupling of agonist‐induced AMPA receptor internalization with receptor recycling
Authors: Liang Feng , Huganir Richard L.
Published: March 2003

Excitatory post‐synaptic currents in the CNS are primarily mediated by α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionic acid (AMPA) receptors in response to glutamate. Internalization of cell‐surface receptors has been shown to be one mechanism by which to control receptor function. To test for agonist control of AMPA receptor plasma membrane expression we used biochemical assays to study AMPA receptor internalization and insertion processes. In heterologous cells, we observed a slow constitutive internalization and a rapid agonist‐induced internalization of AMPA receptors. To our surprise, however, agonist treatment had no effect on the steady‐state levels of AMPA receptors on the cell surface. To examine whether this could be explained by an agonist‐induced increase in the insertion rate of AMPA receptors into the plasma membrane we developed an assay to independently measure receptor insertion. Remarkably, agonist treatment of cells also dramatically increased AMPA receptor plasma membrane insertion rates. In addition, using an assay to measure recycling of internalized pools we found that internalized receptors are rapidly recycled to the cell surface. These results suggest that agonist‐induced receptor internalization is coupled to increases in receptor recycling. This increase in receptor flux through intracellular pools may allow for rapid changes in receptor surface expression by independent regulatory control of internalization and insertion.
Identification and functional characterization of thromboxane A2 receptors in Schwann cells
Authors: Muja Naser , Blackman Samuel C. , Le Breton Guy C. , DeVries George H.
Published: March 2003

Previous reports have demonstrated the presence of functional thromboxane A2 (TP) receptors in astrocytes and oligodendrocytes. In these experiments, the presence and function of TP receptors in primary rat Schwann cells (rSC) and a neurofibrosarcoma‐derived human Schwann cell line (T265) was investigated. Immunocytochemical and immunoblot analyses using polyclonal anti‐TP receptor antibodies demonstrate that both cell types express TP receptors. Treatment with the stable thromboxane A2 mimetic U46619 (10 µm) did not stimulate intracellular calcium mobilization in rSC, whereas T265 cells demonstrated a calcium response that was inhibited by prior treatment with TP receptor antagonists. U46619 also stimulated CREB phosphorylation on Ser133 in T265 cells and, to a lesser extent, in rSC. To identify potential mechanisms of CREB phosphorylation in rSC, we monitored intracellular cAMP levels following U46619 stimulation. Elevated levels of cAMP were detected in both rSC (20‐fold) and T265 (15‐fold) cells. These results demonstrate that TP receptor activation specifically stimulates CREB phosphorylation in T265 cells, possibly by a calcium‐ and/or cAMP‐dependent mechanism. In contrast, TP receptor activation in rSC stimulates increases in cAMP and CREB phosphorylation but does not elicit changes in intracellular calcium.
Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors
Authors: Douhou Aïcha , Troadec Jean‐Denis , Ruberg Merle , Raisman‐Vozari Rita , Michel Patrick P.
Published: March 2003

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20–50 mm). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK‐801 and GYKI‐52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK‐801 or GYKI‐52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK‐801 were mimicked by BAY K‐8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca2+ influx through L‐type calcium channels. Measurement of intracellular calcium revealed that MK‐801 or GYKI‐52466 were required to maintain Ca2+ levels within a trophic range, thus preventing K+‐induced excitotoxic stress and Ca2+ overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.
Transfection‐enforced Bcl‐2 overexpression and an anti‐Parkinson drug, rasagiline, prevent nuclear accumulation of glyceraldehyde‐3‐phosphate dehydrogenase induced by an endogenous dopaminergic neurotoxin, N‐methyl(R)salsolinol
Authors: Maruyama Wakako , Akao Yukihiro , Youdim Moussa B. H. , Davis Bruce A. , Naoi Makoto
Published: March 2003

An endogenous dopaminergic neurotoxin, N‐methyl(R)salsolinol, was found to induce apoptosis in human dopaminergic SH‐SY5Y cells by step‐wise activation of apoptotic cascade; collapse in mitochondrial membrane potential, ΔΨm, activation of caspases, and fragmentation of DNA. Recently, accumulation of gylceraldehyde‐3‐phosphate dehydrogenase (GAPDH) in nuclei was proposed to play an important role in apoptosis. In this paper, involvement of GAPDH in apoptosis induced by N‐methyl(R)salsolinol was studied. The isoquinoline reduced ΔΨm within 3 h, as detected by a fluorescence indicator, JC‐1, then after 16 h incubation, GAPDH accumulated in nuclei by detection with immunostaining. To clarify the role of GAPDH in apoptotic process, a stable cell line of Bcl‐2 overexpressed SH‐SY5Y cells was established. Overexpression of Bcl‐2 prevented the decline in ΔΨm and also apoptotic DNA damage induced by N‐methyl(R)salsolinol. In Bcl‐2 transfected cells, nuclear translocation of GAPDH was also completely suppressed. In addition, a novel antiparkinsonian drug, rasagiline, prevented nuclear accumulation of GAPDH induced by N‐methyl(R)salsolinol in control cells. These results suggest that GAPDH may accumulate in nuclei as a consequence of signal transduction, which is antagonized by anti‐apoptotic Bcl‐2 protein family and rasagiline. The results are discussed in concern to intracellular mechanism underlying anti‐apoptotic function of rasagiline analogues.
Cholesterol depletion by methyl‐β‐cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons
Authors: Shogomori Hidehiko , Futerman Anthony H.
Published: March 2003

We recently demonstrated that although cholera toxin (CT) is found in detergent‐insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft‐independent mechanism. Thus, cholesterol depletion by methyl‐β‐cyclodextrin (MβCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes. In the current study, we analyze which step of CT intracellular transport is inhibited by MβCD. Whereas pre‐incubation with MβCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor‐mediated and fluid phase endocytosis, respectively. Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation. In contrast, MβCD did not affect CT degradation, or CT‐mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus. We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol‐dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol‐independent.
Maturation of rat brain is accompanied by differential expression of the long and short splice variants of Gsα protein: identification of cytosolic forms of Gsα
Authors: Ihnatovych Ivanna , Hejnová Lucie , Koštrnová Alexandra , Mareš Pavel , Svoboda Petr , Novotný Jiří
Published: March 2003

Distribution of the α subunit of the stimulatory G protein (Gsα) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post‐natal development. In parallel, changes in β‐adrenoceptor density and adenylyl cyclase activity were determined. Long (GsαL) and short (GsαS) variants of Gsα were assessed by immunoblotting using specific polyclonal antisera reacting with both Gsα isoforms. Post‐natal development was associated with an increase in the total amount of brain Gsα. GsαL was the dominant isoform of Gsα in the membrane fractions of all studied brain regions and its amount increased markedly between post‐natal day (PD) 1 and 90. The level of membrane‐bound GsαS also elevated during post‐natal development, but more pronounced changes were found in cytosolic GsαS. Although only a small amount of GsαS (much smaller than GsαL) was detected among soluble proteins shortly after birth, GsαS prevailed over GsαL at PD90. The GsαL/GsαS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the GsαL/GsαS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane–cytosol balance of Gsα proteins alters dramatically during the course of brain development. Both GsαL and GsαS were expressed in a region‐ and age‐specific manner, which suggests different roles in the maturation of the brain tissue. A cyc− reconstitutive assay of cytosolic Gsα indicated that only ≈ 20% of this protein was functional, compared with membrane‐bound Gsα, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of β‐adrenoceptors increased sharply during early post‐natal development but only slightly in adulthood, and both GTP‐ and isoproterenol‐stimulated adenylate cyclase activity reached peak values around PD12.
Effects of threo‐β‐hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5)
Authors: Shigeri Yasushi , Shimamoto Keiko , Yasuda‐Kamatani Yoshimi , Seal Rebecca P. , Yumoto Noboru , Nakajima Terumi , Amara Susan G.
Published: March 2003

d,lthreo‐β‐Benzyloxyaspartate (d,l‐TBOA), an analog of threo‐β‐hydroxyaspartate (THA) possessing a bulky substituent, is a potent non‐transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while lthreo‐β‐methoxyaspartate (l‐TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4‐expressing oocytes, d,l‐TBOA acted as a non‐transportable blocker, while l‐TMOA like d,l‐THA was a competitive substrate. In contrast, d,l‐THA, d,l‐TBOA and l‐TMOA all strongly attenuated the glutamate‐induced currents generated by EAAT5. Among them, l‐TMOA showed the most potent inhibitory action. Moreover, d,l‐THA, d,l‐TBOA and l‐TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that d,l‐TBOA and l‐TMOA, as well as d,l‐THA, not only act as non‐transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate‐gated chloride channel.
A comparison of morphine‐induced locomotor activity and mesolimbic dopamine release in C57BL6, 129Sv and DBA2 mice
Authors: Murphy Niall P. , Lam Hoa A. , Maidment Nigel T.
Published: March 2003

Inbred mouse strains show marked variations in morphine‐induced locomotion and reward behaviors. As increases in mesolimbic dopamine release and locomotion have been implicated as being critical aspects of drug‐seeking and reward‐related behaviors, the present study sought to determine the relationship between morphine‐induced changes in locomotion and mesolimbic dopamine release. Freely moving microdialysis of the ventral striatum was performed in mouse strains chosen on the basis of their documented differences in locomotor and reward response to morphine (C57BL6 and DBA2) and use in the production of genetically modified mice (129Sv). Both C57BL6 and 129Sv mice showed significant increases in locomotion and ventral striatal extracellular dopamine levels following subcutaneous morphine administration (3 mg/kg), with the former strain showing the largest increase in both parameters. Ventral striatal extracellular DA levels increased in DBA2 mice to a similar extent as 129Sv mice following morphine administration, despite this strain showing no locomotor response. Intra‐strain analysis found no correlation between morphine‐induced locomotion and mesolimbic dopamine release in any of the strains studied. Thus, no universal relationship between morphine‐induced mesolimbic dopamine release and locomotion exists between, and particularly within, inbred mouse strains. Furthermore, morphine‐induced increases in mesolimbic activity correlate negatively with the rewarding potential of morphine described in previously reported conditioned place preference studies.
Non‐MAO A binding of clorgyline in white matter in human brain
Authors: Fowler J. S. , Logan J. , Ding Y.‐S. , Franceschi D. , Wang G.‐J. , Volkow N. D. , Pappas N. , Schlyer D. , Gatley S. J. , Alexoff D. , Felder C. , Biegon A. , Zhu W.
Published: March 2003

Clorgyline is an irreversible inhibitor of monoamine oxidase (MAO A) which has been labeled with carbon‐11 (C‐11) and used to measure human brain MAO A with positron emission tomography (PET). In this study we compared [11C]clorgyline and deuterium‐substituted [11C]clorgyline ([11C]clorgyline‐D2) to better understand the molecular link between [11C]clorgyline binding and MAO A. In PET studies of five normal healthy volunteers scanned with [11C]clorgyline and [11C]clorgyline‐D2 2 h apart, deuterium substitution generally produced the expected reductions in the brain uptake of [11C]clorgyline. However, the reduction was not uniform with the C‐11 binding in white matter being significantly less sensitive to deuterium substitution than other brain regions. The percentages of the total binding attributable to MAO A is largest for the thalamus and smallest for the white matter and this is clearly seen in PET images with [11C]clorgyline‐D2. Thus deuterium‐substituted [11C]clorgyline selectively reduces the MAO A binding component of clorgyline in the human brain revealing non‐MAO A binding which is most apparent in the white matter. The characterization of the non‐MAO A binding component of this widely used MAO A inhibitor merits further investigation.
Ethanol potentiates the function of the human dopamine transporter expressed inXenopusoocytes
Authors: Mayfield R. Dayne , Maiya Rajani , Keller Denise , Zahniser Nancy R.
Published: March 2003

Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed inXenopusoocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nm) accumulation into DAT‐expressing oocytes increased significantly in response to ethanol (10 min; 10–100 mm). In two‐electrode voltage‐clamp experiments, DAT‐mediated currents were also enhanced significantly by ethanol (10–100 mm). The magnitude of the ethanol‐induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nm) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT‐mediated substrate translocation and substrate‐associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface.
Functional significance of nitric oxide in ionomycin‐evoked [3H]GABA release from mouse cerebral cortical neurons
Authors: Katsura Masashi , Shuto Keijiro , Mohri Yutaka , Shigeto Makoto , Ohkuma Seitaro
Published: March 2003

We investigated a role of nitric oxide (NO) on ionomycin‐evoked [3H]GABA release using mouse cerebral cortical neurons. Ionomycin dose‐dependently released [3H]GABA up to 1 µm. The extent of the release by 0.1 µmionomycin was in a range similar to that by 30 mmKCl. The ionomycin (0.1 µm)‐evoked [3H]GABA release was dose‐dependently inhibited by NO synthase inhibitors and hemoglobin, indicating that the ionomycin‐evoked [3H]GABA release is mediated through NO formation. The inhibition of cGMP formation by 1H‐[1,2,4] oxodizao [4,3‐a] quinoxalin‐1‐one (ODQ), a selective inhibitor for NO‐sensitive guanylate cyclase, showed no affects on the ionomycin‐evoked [3H]GABA release. Tetrodotoxin and dibucaine significantly suppressed the ionomycin‐evoked [3H]GABA release and ionomycin increased fluorescence intensity of bis‐oxonol, suggesting the involvement of membrane depolarization in this release. The ionomycin‐evoked [3H]GABA release was maximally reduced by about 50% by GABA uptake inhibitors. The concomitant presence of nifedipine and ω‐agatoxin VIA (ω‐ATX), inhibitors for L‐ and P/Q‐type voltage‐dependent calcium channels, respectively, caused the reduction in the ionomycin‐evoked release by about 50%. The simultaneous addition of nifedipine, ω‐ATX and nipecotic acid completely abolished the release. Although ionomycin released glutamate, (+)‐5‐methyl‐1‐,11‐dihydro‐5H‐dibenzo‐[a,d]cycloheptan‐5,10‐imine (MK‐801) and 6,7‐ dinitroquinoxaline‐2,3‐dione (DNQX) showed no effects on the ionomycin‐induced [3H]GABA release. Based on these results, it is concluded that NO formed by ionomycin plays a critical role in ionomycin‐evoked [3H]GABA release from the neurons.
An oxidative stress‐mediated positive‐feedback iron uptake loop in neuronal cells
Authors: Núñez‐Millacura Claudia , Tapia Victoria , Muñoz Patricia , Maccioni Ricardo B. , Núñez Marco T.
Published: March 2003

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 µm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 µm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 µmwas functional as it correlated with increased55Fe uptake. The increase in IRP1 activity was mediated by oxidative‐stress as it was largely abolished byN‐acetyl‐l‐cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.
Acute administration of cocaine regulates the phosphorylation of serine‐19, ‐31 and ‐40 in tyrosine hydroxylase
Authors: Jedynak Jakub P. , Ali Syed F. , Haycock John W. , Hope Bruce T.
Published: March 2003

Acute cocaine can inhibit catecholamine biosynthesis by regulating the enzymatic activity of tyrosine hydroxylase via alterations in the phosphorylation state of the enzyme. The mechanisms underlying acute cocaine‐dependent regulation of tyrosine hydroxylase phosphorylation have not been determined. In this study, 0, 15 or 30 mg/kg cocaine was administered intraperitoneally to rats and the phosphorylation state of tyrosine hydroxylase in the brain was examined using antibodies specific for the phosphorylated forms of serine‐19, ‐31 and ‐40 in tyrosine hydroxylase. In the caudate and nucleus accumbens, cocaine dose‐dependently decreased the levels of phosphorylated serine‐19, ‐31 and ‐40. In the ventral tegmental area, the levels of phosphorylated serine‐19, but not serine‐31 and ‐40, were decreased by 15 and 30 mg/kg cocaine. In the amygdala, the levels of phosphorylated serine‐19, but not serine‐31 or ‐40, were decreased. The functional effects of these alterations in phosphorylation state were assessed by measuring tyrosine hydroxylase activityin vivo(accumulation of DOPA after administration of the decarboxylase inhibitor NSD‐1015). Acute administration of 30 mg/kg cocaine significantly decreasedl‐DOPA production in caudate and accumbens but not in amygdala. These data suggest that the phosphorylation of serine‐31 or ‐40, but not serine‐19, is involved in the regulation of tyrosine hydroxylase activity by acute cocaine.
Prominent Narp expression in projection pathways and terminal fields
Authors: Reti Irving M. , Reddy Radhika , Worley Paul F. , Baraban Jay M.
Published: March 2003

Narp (neuronal activity regulated pentraxin) is a secreted immediate early gene product that is induced by synaptic activity. Recent studies have indicated that Narp may be an extracellular aggregating factor for AMPA receptors. Immunohistochemical studies have revealed prominent expression of Narp in the mossy fiber pathway of the dentate gyrus, suggesting it may be released pre‐synaptically. However, invitro studies using recombinant Narp indicate that Narp may act when expressed by either pre‐ or post‐synaptic elements. To help define Narp's mode of action, we have examined its localization in the habenula‐interpeduncular pathway which also displays robust Narp expression. Focusing on this pathway as well as hippocampal and cortical Narp expression, we found prominent Narp staining in projection pathways and terminal fields. In contrast, Narp expression in dendrites was minimal in these neuronal populations. These findings indicate that, under physiological conditions, Narp is targeted to the synapse from pre‐ rather than post‐synaptic elements. Our results also suggest that future studies focusing on these projection pathways that express high levels of Narp, in vivo, may help to understand the regulation and function of endogenous Narp.
Failure of the interaction between presenilin 1 and the substrate of γ‐secretase to produce Aβ in insect cells
Authors: Pitsi Didier , Kienlen‐Campard Pascal , Octave Jean‐Noël
Published: March 2003

Aggregates of β‐amyloid peptide (Aβ) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Aβ results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by β‐ and γ‐secretase activities. If β‐secretase has recently been identified and termed BACE, the identity of γ‐secretase is still obscure. Studies with knock‐out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the γ‐secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous β‐ and/or γ‐secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected β‐secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of γ‐secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Aβ. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the γ‐secretase.
Adenosine A2a receptor‐mediated inhibition of rod opsin mRNA expression in tiger salamander
Authors: Alfinito Peter D. , Alli Roshni , Townes‐Anderson Ellen
Published: March 2003

The neuromodulator adenosine mediates dark‐adaptive changes in retinal photoreceptors through A2a receptors. In cold‐blooded vertebrates, opsin mRNA expression is lower at night than during the day. In the present study, we tested whether adenosine could inhibit opsin mRNA expression in cultured rod cells and if endogenous adenosine acts to suppress opsin mRNA in the intact retina at night. Semi‐quantitative in situ hybridization showed that treatment with 100 nm of the A2a/A2b agonist N 6‐[2‐(3,5‐dimethoxyphenyl)‐2‐(2‐methylphenyl)‐ethyl]adenosine (DPMA) reduced opsin mRNA 41% in cultured rod cells. The effect of DPMA was blocked by 10 µm of the A2a antagonist 8‐(3‐chlorostyryl)caffeine (CSC) but not by 10 µm of the A2b antagonist alloxazine. One micromolar adenosine alone had no effect on opsin mRNA. However, in the presence of the adenosine deaminase inhibitor erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine hydrochloride (EHNA), 1 µm adenosine reduced opsin mRNA 61%. EHNA alone reduced opsin mRNA by 26%. Consistent with an A2a receptor mechanism, 100 nm forskolin (adenylate cyclase agonist) decreased opsin mRNA 34%. Finally, northern blots showed that intravitreal injection of 10 µm CSC at night increased opsin I mRNA 38%. Thus, endogenous adenosine suppresses rod opsin I mRNA expression at night; in vitro results indicate this reduction occurs through A2a‐like receptor binding and stimulation of adenylate cyclase activity.
Urotensin‐II regulates intracellular calcium in dissociated rat spinal cord neurons
Authors: Filipeanu Catalin M. , Brailoiu Eugen , Le Dun Siok , Dun Nae J.
Published: March 2003

Urotensin‐II (U‐II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U‐II on [Ca2+]i was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase‐positive and had morphological features of motoneurons. U‐II induced [Ca2+]i increases in these neurons with a threshold of 10−9 m, and a maximal effect at 10−6 m with an estimated EC50 of 6.2 × 10−9 m. The [Ca2+]i increase induced by U‐II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+‐free medium. Omega‐conotoxin GVIA (10−7 m), a N‐type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega‐conotoxin GVIIC (10−7 m) and the l‐type Ca2+ channel blocker, verapamil (10−5 m) had minimal effects. Down‐regulation of protein kinase C by 4‐α‐phorbol 12‐myristate 13‐acetate (10−6 m) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10−6 m) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10−6 m) or Rp‐8‐pCPT‐cGMPS (3 × 10−5 m) did not modify U‐II‐induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10−6 m) and Rp‐cAMPS (3 × 10−5 m) reduced the response to 25 ± 3% and 42 ± 8%, respectively. Present results demonstrate that U‐II modulates [Ca2+]i in rat spinal cord neurons via protein kinase A cascade.
Microarray analysis of selected genes in neural stem and progenitor cells
Authors: Luo Yongquan , Cai Jingli , Liu Ying , Xue Haipeng , Chrest Francis J. , Wersto Robert P. , Rao Mahendra
Published: March 2003

To access and compare gene expression in fetal neuroepithelial cells (NEPs) and progenitor cells, we have used microarrays containing approximately 500 known genes related to cell cycle regulation, apoptosis, growth and differentiation. We have identified 152 genes that are expressed in NEPs and 209 genes expressed by progenitor cells. The majority of genes (141) detected in NEPs are also present in progenitor populations. There are 68 genes specifically expressed in progenitors with little or no expression in NEPs, and a few genes that appear to be present exclusively in NEPs. Using cell sorting, RT–PCR, in situ hybridization or immunocytochemistry, we have examined the segregation of expression to neuronal and glial progenitors, and identified several that appeared to be enriched in neuronal (e.g. CDK5, neuropilin, EphrinB2, FGF11) or glial (e.g. CXCR4, RhoC, CD44, tenascin C) precursors. Our data provide a first report of gene expression profiles of neural stem and progenitor cells at early stages of development, and provide evidence for the potential roles of specific cell cycle regulators, chemokines, cytokines and extracellular matrix molecules in neural development and lineage segregation.
Mutant prion protein‐mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity
Authors: Gu Yaping , Verghese Susamma , Mishra Ravi Shankar , Xu Xeumin , Shi Yongchang , Singh Neena
Published: March 2003

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC–GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC–GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C‐terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.