bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct signal transduction pathways for GABA‐induced GABAA receptor down‐regulation and uncoupling in neuronal culture: a role for voltage‐gated calcium channels
Authors: Lyons Helen R. , Land Michele B. , Gibbs Terrell T. , Farb David H.
Published: March 2003

Changes in GABA receptor (GABAAR) gene expression are detected in animal models of epilepsy, anxiety and in post‐mortem schizophrenic brain, suggesting a role for GABAAR regulation in neurological disorders. Persistent (48 h) exposure of brain neurons in culture to GABA results in down‐regulation of GABAAR number and uncoupling of GABA and benzodiazepine (BZD) binding sites. Given the central role of GABAARs in fast inhibitory synaptic transmission, GABAAR down‐regulation and uncoupling are potentially important mechanisms of regulating neuronal excitability, yet the molecular mechanisms remain unknown. In this report we show that treatment of brain neurons in culture with tetrodotoxin, glutamate receptor antagonists, or depolarization with 25 mm K+ fails to alter GABAAR number or coupling. Changes in neuronal activity or membrane potential are therefore not sufficient to induce either GABAAR down‐regulation or uncoupling. Nifedipine, a voltage‐gated Ca2+ channel (VGCC) blocker, inhibits both GABA‐induced increases in [Ca2+]i and GABAAR down‐regulation, suggesting that VGCC activation is required for GABAAR down‐regulation. Depolarization with 25 mm K+ produces a sustained increase in intracellular [Ca2+] without causing GABAAR down‐regulation, suggesting that activation of VGCCs is not sufficient to produce GABAAR down‐regulation. In contrast to GABAAR down‐regulation, nifedipine and 25 mm K+ fail to inhibit GABA‐induced uncoupling, demonstrating that GABA‐induced GABAAR down‐regulation and uncoupling are mediated by independent molecular events. Therefore, GABAAR activation initiates at least two distinct signal transduction pathways, one of which involves elevation of intracellular [Ca2+] through VGCCs.
Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [3H]azidoneonicotinoid
Authors: Tomizawa Motohiro , Wen Zhimou , Chin Hsiao‐Ling , Morimoto Hiromi , Kayser Hartmut , Casida John E.
Published: March 2003

The nicotinic acetylcholine receptor (nAChR) is a ligand‐gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5‐azido‐6‐chloropyridin‐3‐yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non‐azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [3H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [3H]AzNN binds to a single high‐affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [3H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to ∼45 and ∼56 kDa, respectively. The photoaffinity‐labeled receptor in both Drosophila and Musca has imidacloprid‐ and nicotine‐sensitive profiles and migrates at ∼66 kDa. These photoaffinity‐labeled polypeptides are considered to be the insecticide‐binding subunits of native insect nAChRs.
Increased expression of the neuropeptide Y receptor Y1 gene in the medial amygdala of transgenic mice induced by long‐term treatment with progesterone or allopregnanolone
Authors: Ferrara Gianna , Serra Mariangela , Zammaretti Francesca , Pisu Maria Giuseppina , Panzica GianCarlo , Biggio Giovanni , Eva Carola
Published: March 2003

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABAA receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y1R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y1 receptor for NPY linked to the lacZ gene, we previously showed that long‐term treatment with benzodiazepine receptor ligands modulates Y1 receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y1R/LacZ transgene expression, as determined by quantitative histochemical analysis of β‐galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and β‐galactosidase expression in the medial amygdala. Finasteride, a 5α‐reductase inhibitor, prevented both of these effects. Long‐term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected β‐galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y1 receptor gene expression through modulation of GABAA receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.
Evaluation of neurotoxicity of TIQ and MPTP and of parkinsonism‐preventing effect of 1‐MeTIQ by in vivo measurement of pre‐synaptic dopamine transporters and post‐synaptic dopamine D2 receptors in the mouse striatum
Authors: Ishiwata Kiichi , Koyanagi Yasuhiro , Abe Kenji , Kawamura Kazunori , Taguchi Kyoji , Saitoh Toshiaki , Toda Jun , Senda Michio , Sano Takehiro
Published: March 2003

Parkinsonism‐inducing neurotoxicity of 1,2,3,4‐tetrahydroisoquinoline (TIQ), as contrasted to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), and parkinsonism‐preventing effect of 1‐methyl‐1,2,3,4‐tetrahydroisoquinoline (1‐MeTIQ) have been investigated in mice by measuring their effects on the in vivo binding of radioligand to pre‐synaptic dopamine transporters (DATs) or to dopamine D2 receptors (D2R) in the striatum. A significant reduction of the ligand‐DATs binding was found in the mice treated with MPTP, but not with TIQ, under the dosage inducing behavioral abnormality and loss of tyrosine hydroxylase‐positive cells in the substantia nigra. A slight decrease in the ligand‐DATs binding was observed in the mice given a larger dose of TIQ. Compensatory up‐regulation in the post‐synaptic D2Rs was found in the MPTP‐treated mice. Pre‐treatment with (S)‐enantiomer, but not (R)‐enantiomer, of 1‐MeTIQ prevented the degeneration of DATs to some extent. We concluded that the TIQ‐induced parkinsonism model is different from the MPTP‐induced model as evaluated by the radioligand‐DATs binding and that (S)‐1‐MeTIQ has a preventing effect for the degeneration of the DATs to a certain extent.
Selective age‐related changes in the PKC‐sensitive, calmodulin‐binding protein, neurogranin, in the mouse brain
Authors: Mons N. , Enderlin V. , Jaffard R. , Higueret P.
Published: March 2003

Brain ageing is associated with a dysregulation of intracellular calcium (Ca2+) homeostasis which leads to deficits in Ca2+‐dependent signalling pathways and altered neuronal functions. Given the crucial role of neurogranin/RC3 (Ng) in the post‐synaptic regulation of Ca2+ and calmodulin levels, age‐dependent changes in the levels of Ng mRNA and protein expression were analysed in 3, 12, 24 and 31‐month‐old mouse brains. Ageing produced significant decreases in Ng mRNA expression in the dorsal hippocampal subfields, retrosplenial and primary motor cortices, whereas no reliable changes were seen in any other cortical regions examined. Western blot indicated that Ng protein expression was also down‐regulated in the ageing mouse brain. Analysis of Ng immunoreactivity in both hippocampal CA1 and retrosplenial areas indicated that Ng protein in aged mice decreased predominantly in the dendritic segments of pyramidal neurones. These data suggest that age‐related changes of post‐synaptic Ng in selected brain areas, and particularly in hippocampus, may contribute to altered Ca2+/calmodulin‐signalling pathways and to region‐specific impairments of synaptic plasticity and cognitive decline.
Long‐term behavioral and neurochemical effects of chronic stress exposure in rats
Authors: Mangiavacchi Simona , Masi Flavio , Scheggi Simona , Leggio Benedetta , De Montis M. Graziella , Gambarana Carla
Published: March 2003

Rats exposed to acute unavoidable stress develop a deficit in escaping avoidable aversive stimuli that lasts as long as unavoidable stress exposure is repeated. A 3‐week exposure to unavoidable stress also reduces dopamine (DA) output in the nucleus accumbens shell (NAcS). This study showed that a 7‐day exposure to unavoidable stress induced in rats an escape deficit and a decrease in extraneuronal DA basal concentration in the NAcS. Moreover, animals had reduced DA and serotonin (5‐HT) accumulation after cocaine administration in the medial pre‐frontal cortex (mPFC) and NAcS, compared with control animals. After a 3‐week exposure to unavoidable stress, escape deficit and reduced DA output in the NAcS were still significant at day 14 after the last stress administration. In the mPFC we observed: (i) a short‐term reduction in DA basal levels that was back to control values at day 14; (ii) a decrease in DA accumulation at day 3 followed by a significant increase beyond control values at day 14; (iii) a significant reduction in 5‐HT extraneuronal basal levels at day 3, but not at day 14. Finally, a significant decrease in 5‐HT accumulation following cocaine administration was present in the NAcS and mPFC at day 3, but not at day 14. In conclusion, a long‐term stress exposure induced long‐lasting behavioral sequelae associated with reproducible neurochemical modifications.
Co‐administration of IGF‐I and glycosaminoglycans greatly delays motor neurone disease and affects IGF‐I expression in the wobbler mouse: a long‐term study
Authors: Gorio Alfredo , Lesma Elena , Madaschi Laura , Di Giulio Anna Maria
Published: March 2003

The study on wobbler mouse has shown that the combined treatment with low doses of glycosaminoglycans (GAGs) and insulin‐like growth factor‐I (IGF‐I) fully prevented motor neurone death and forelimb impairment up to 9–12 weeks of a mouse's life. The effect was accompanied by the prevention of the early hypertrophy of wobbler neurones, an effect likely due to the promotion of neuronal survival. At the 18th week, wobbler mice treated with IGF‐I + GAGs still showed significantly improved forelimb function, reduced muscle atrophy and a higher number of cervical motor neurones. IGF‐I alone and GAGs alone were active up to the 3rd week of treatment; thereafter the beneficial effects of single treatments decreased drastically. GAGs and IGF‐I treatments also affected IGF‐I plasma and muscle levels. In wobbler mice there was a progressive reduction in IGF‐I plasma levels that was prevented by IGF‐I or GAGs alone and greatly increased, even above heterozygote levels, by the combination treatment. Such a powerful increase was correlated by a small enhancement in insulin‐like growth factor binding protein‐3 (IGFBP‐3) plasma levels, while treatment with IGF‐I alone affected very significantly both IGFBP‐1 and IGFBP‐3. Co‐treatment also prevented the decrease in IGF‐I content observed in vehicle‐treated wobbler mice forelimb muscles.
Expression of the neonatal Fc receptor (FcRn) at the blood–brain barrier
Authors: Schlachetzki Felix , Zhu Chunni , Pardridge William M.
Published: March 2003

The blood–brain barrier (BBB) restricts transport of immunoglobulin G (IgG) in the blood to brain direction. However, IgG undergoes rapid efflux in the brain to blood direction via reverse transcytosis across the BBB after direct intracerebral injection. This BBB IgG transport system has the characteristics of an Fc receptor (FcR), but there is no molecular information on the putative BBB FcR. The present study uses confocal microscopy and an antibody to the rat neonatal FcR (FcRn), and demonstrates the expression of the FcRn at the brain microvasculature and choroid plexus epithelium. Co‐localization with the Glut1 glucose transporter indicates the brain microvascular FcRn is expressed in the capillary endothelium. The capillary endothelial FcRn may mediate the ‘reverse transcytosis’ of IgG in the brain to blood direction.
Mass‐spectrometric analysis of myelin proteolipids reveals new features of this family of palmitoylated membrane proteins
Authors: Bizzozero Oscar A. , Malkoski Steve P. , Mobarak Charlotte , Bixler Heather A. , Evans James E.
Published: March 2003

In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM‐20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size‐exclusion and ion‐exchange chromatography in organic solvents. Matrix‐assisted laser desorption ionization‐time of flight‐mass spectrometry (MALDI–TOF–MS) of PLP and DM‐20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester‐bound fatty acids. Electrospray ionization‐MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post‐translational modification of PLP, and that the majority of PLP and DM‐20 molecules in the CNS are fully acylated. A series of myelin‐associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N‐ and C‐terminal sequencing, tryptic digestion and peptide mapping by MALDI–TOF–MS. The results clearly showed that these polypeptides correspond to the N‐terminal region (residues 1–105/112) and C‐terminal region (residues 113/131–276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid‐long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM‐20, and are differentially distributed across the CNS.
Inhibitors of nitric oxide synthase attenuate nerve growth factor‐mediated increases in choline acetyltransferase expression in PC12 cells
Authors: Kalisch Bettina E. , Bock Nicholas A. , Davis Wanda L. , Rylett R. Jane
Published: March 2003

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF‐mediated neurotrophic responses. To investigate the role of NO in NGF‐activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor l‐NAME (N ω‐nitro‐l‐arginine methylester) or the inducible NOS selective inhibitor MIU (s‐methylisothiourea), and the effect on NGF‐stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady‐state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, l‐NAME blocked neurite outgrowth in nerve growth factor (NGF)‐treated PC12 cells. Both l‐NAME and MIU attenuated NGF‐mediated increases in choline transferase (ChAT)‐specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.
Protein synthesis in synaptosomes: a proteomics analysis
Authors: Jiménez C. R. , Eyman M. , Lavina Z. Scotto , Gioio A. , Li K. W. , Van Der Schors R. C. , Geraerts W. P. M. , Giuditta A. , Kaplan B. B. , Van Minnen J.
Published: March 2003

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre‐synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [35S]methionine and applied two‐dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two‐dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver‐stained gels that were run in parallel. In‐gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear‐encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre‐synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.
The RNA‐binding protein Staufen from rat brain interacts with protein phosphatase‐1
Authors: Monshausen Michaela , Rehbein Monika , Richter Dietmar , Kindler Stefan
Published: March 2003

In mammalian neurones, homologues of the Drosophila RNA‐binding protein Staufen are part of ribonucleoprotein complexes that move bidirectionally along dendritic microtubules and appear to regulate mRNA translocation and translation. In this study, putative components of Staufen granules were identified in a yeast two‐hybrid screen of a rat brain cDNA library with a rat Staufen bait. Protein phosphatase‐1 was found as an interacting partner. Binding appears to be mediated by a five amino acid residue sequence motif (R‐K‐V‐T‐F) in Staufen that is conserved in a number of proteins interacting with the phosphatase. A two amino acid residue mutation within this motif (R‐K‐V‐G‐A) disrupted the interaction. A cytoplasmic interaction of both proteins was shown by coimmunoprecipitation of rat Staufen and protein phosphatase‐1 from the cytoplasm of transfected cells and rat brain homogenates. In mammalian brain, the phosphatase represents the first described endogenous interaction partner of Staufen. In primary hippocampal neurones, both proteins partially colocalize in somata and neuronal processes. Staufen does not modulate the in vitro protein phosphatase activity. These findings show that protein phosphatase‐1 is a native component of Staufen particles. Cellular functions of Staufen may be regulated via phosphorylation or Staufen may recruite the phosphatase into specific ribonucleoprotein complexes.
ProSAP/Shank proteins – a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease
Authors: Boeckers Tobias M. , Bockmann Jürgen , Kreutz Michael R. , Gundelfinger Eckart D.
Published: March 2003

The postsynaptic density (PSD) is a specialized electron‐dense structure underneath the postsynaptic plasmamembrane of excitatory synapses. It is thought to anchor and cluster glutamate receptors exactly opposite to the presynaptic neurotransmitter release site. Various efforts to study the molecular structure of the PSD identified several new proteins including membrane receptors, cell adhesion molecules, components of signalling cascades, cytoskeletal elements and adaptor proteins with scaffolding functions to interconnect these PSD components. The characterization of a novel adaptor protein family, the ProSAPs or Shanks, sheds new light on the basic structural organization of the PSD. ProSAPs/Shanks are multidomain proteins that interact directly or indirectly with receptors of the postsynaptic membrane including NMDA‐type and metabotropic glutamate receptors, and the actin‐based cytoskeleton. These interactions suggest that ProSAP/Shanks may be important scaffolding molecules of the PSD with a crucial role in the assembly of the PSD during synaptogenesis, in synaptic plasticity and in the regulation of dendritic spine morphology. Moreover the analysis of a patient with 22q13.3 distal deletion syndrome revealed a balanced translocation with a breakpoint in the human ProSAP2/Shank3 gene. This ProSAP2/Shank3 haploinsufficiency may cause a syndrome that is characterized by severe expressive language delay, mild mental retardation and minor facial dysmorphisms.
Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain
Authors: Toneff Thomas , Mende‐Mueller Liane , Wu Ying , Hwang Shin‐Rong , Bundey Richard , Thompson Leslie M. , Chesselet Marie‐Francoise , Hook Vivian
Published: March 2003

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope‐specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH2‐ and COOH‐terminal htt fragments of 20–100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63–64 kDa htt fragment detected by the NH2‐domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60–61 kDa band (detected by the NH2‐domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63–64 kDa and 60–61 kDa NH2‐domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66‐kDa, instead of the 63–64 kDa N‐domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH2‐ and COOH‐terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH2‐terminal htt fragments [detected with anti(1–17) serum] compared to controls. Cortex from HD and control brains showed similar NH2‐terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH2‐terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48‐kDa COOH‐terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH2‐terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.
Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis
Authors: Han Xianlin , M. Holtzman David , W. McKeel Daniel , Kelley John , Morris John C.
Published: March 2003

In addition to pathology in the gray matter, there are also abnormalities in the white matter in Alzheimer's disease (AD). Sulfatide species are a class of myelin‐specific sphingolipids and are involved in certain diseases of the central nervous system. To assess whether sulfatide content in gray and white matter in human subjects is associated with both the presence of Alzheimer's disease (AD) pathology as well as the stage of dementia, we analyzed the sulfatide content of brain tissue lipid extracts by electrospray ionization mass spectrometry from 22 subjects whose cognitive status at time of death varied from no dementia to very severe dementia. All subjects with dementia had AD pathology. The results demonstrate that: (i) sulfatides were depleted up to 93% in gray matter and up to 58% in white matter from all examined brain regions from AD subjects with very mild dementia, whereas all other major classes of lipid (except plasmalogen) in these subjects were not altered in comparison to those from age‐matched subjects with no dementia; (ii) there was no apparent deficiency in the biosynthesis of sulfatides in very mild AD subjects as characterized by the examination of galactocerebroside sulfotransferase activities in post‐mortem brain tissues; (iii) the content of ceramides (a class of potential degradation products of sulfatides) was elevated more than three‐fold in white matter and peaked at the stage of very mild dementia. The findings demonstrate that a marked decrease in sulfatides is associated with AD pathology even in subjects with very mild dementia and that these changes may be linked with early events in the pathological process of AD.
Transient changes in mesolimbic dopamine and their association with ‘reward’
Authors: Wightman R. Mark , Robinson Donita L.
Published: March 2003

Mesolimbic dopaminergic neurons modulate complex circuitry in the ventral forebrain involved in reward processing, although the precise function of the dopaminergic input is debated. Electrophysiological measurements have revealed that mesolimbic dopaminergic neurons can fire in either tonic or phasic modes, and that phasic firing accompanies the alerting or anticipatory phases of reward. However, the neurochemical relevance of this rapid neuronal discharge within the reward processing circuitry is not yet clear, in part because of difficulty in interpretation of extracellular dopamine measurements. Herein, the nature of the information provided by different neurochemical techniques is critically discussed. Classical methods of monitoring dopamine reveal changes in extracellular dopamine resulting from tonic neuronal activity, but do not have the temporal resolution to distinguish concentration transients. However, recent advances in dopamine sensors now enable transient dopamine concentrations resulting from phasic firing to be positively identified and followed on a physiologically relevant timescale. This has enabled demonstrations of discrete, phasic dopamine signals accompanying rewarding or alerting stimuli. Thus, enhanced dopamine release at terminals appears to be coincident with phasic electrical activity at cell bodies. These accumulating data promise to help unravel the precise role of phasic dopamine transmission in reward processing.
The dopamine D1 receptor is a critical mediator for cocaine‐induced gene expression
Authors: Zhang Dongsheng , Zhang Lu , Lou Dan Wen , Nakabeppu Yusaku , Zhang Jianhua , Xu Ming
Published: March 2003

The dopamine D1 receptor plays a major role in mediating behavioral responses to cocaine administration. The time course for the acquisition and the relative stability for the expression of behavioral responses suggest the involvement of enduring neuroadaptations in response to repeated cocaine exposure. Changes in gene expression through the D1 receptors may accompany and mediate the development of such neuroadaptations to repeated cocaine stimulation. To test this possibility, we systematically compared the expression of the fos and Jun family immediate early genes in the nucleus accumbens and caudoputamen in D1 receptor mutant and wild‐type control mice after acute and repeated cocaine exposure. Moreover, we compared the expression of three molecules that have been implicated in mediating the actions of cocaine, Gαolf, β‐catenin and brain‐derived neurotrophic factor, in the two groups of mice before and after cocaine administration. We found that there is a lack of induction of c‐Fos, FosB, Fra‐2 and JunB by acute cocaine exposure, and of ΔFosB by repeated cocaine administration in both the NAc and CPu of D1 receptor mutant mice compared with wild‐type control mice. Moreover, the D1 receptor is differentially required for mediating Gαolf, β‐catenin and BDNF expression in the NAc and CPu upon cocaine exposure. These results suggest that the D1 receptor is a critical mediator for cocaine‐induced expression of these genes.
DLGdifferentially localizes Shaker K+‐channels in the central nervous system and retina of Drosophila
Authors: Ruiz‐Cañada C. , Koh Y. H. , Budnik V. , Tejedor F. J.
Published: March 2003

Subcellular localization of ion channels is crucial for the transmission of electrical signals in the nervous system. Here we show that Discs‐Large (DLG), a member of the MAGUK (membrane‐associated guanylate kinases) family in Drosophila, co‐localizes with Shaker potassium channels (Sh Kch) in most synaptic areas of the adult brain and in the outer membrane of photoreceptors. However, DLG is absent from axonal tracts in which Sh channels are concentrated. Truncation of the C‐terminal of Sh (including the PDZ binding site) disturbs its pattern of distribution in both CNS and retina, while truncation of the guanylate kinase/C‐terminal domain of DLG induces ectopic localization of these channels to neuronal somata in the CNS, but does not alter the distribution of channels in photoreceptors. Immunocytochemical, membrane fractionation and detergent solubilization analysis indicate that the C‐terminal of Sh Kch is required for proper trafficking to its final destination. Thus, several major conclusions emerge from this study. First, DLG plays a major role in the localization of Shchannels in the CNS and retina. Second, localization of DLG in photoreceptors but not in the CNS seems to depend on its interaction with Sh. Third, the guanylate kinase/C‐terminal domain of DLG is involved in the trafficking of Shaker channels but not of DLG in the CNS. Fourth, different mechanisms for the localization of Sh Kch operate in different cell types.
Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase‐related protein 2, α‐enolase and heat shock cognate 71
Authors: Castegna Alessandra , Aksenov Michael , Thongboonkerd Visith , Klein Jon B. , Pierce William M. , Booze Rosemarie , Markesbery William R. , Butterfield D. Allan
Published: March 2003

Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation‐related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox‐balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for labor‐intensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy‐terminal hydrolase L−1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP‐2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme α‐enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC‐71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.
Selective reduction by isolation rearing of 5‐HT1A receptor‐mediated dopamine release in vivo in the frontal cortex of mice
Authors: Ago Y. , Sakaue M. , Baba A. , Matsuda T.
Published: March 2003

Serotonin (5‐HT)1A receptors modulate in vivo release of brain monoaminergic neurotransmitters which may be involved in isolation‐induced aggressive behavior. The present study examined the effect of isolation rearing on the 5‐HT1A receptor‐mediated modulation of dopamine (DA), 5‐HT and noradrenaline (NA) release in the frontal cortex of mice. The selective 5‐HT1A receptor agonist (S)‐5‐{3‐[(1,4‐benzodioxan‐2‐ylmethyl)amino]propoxy}‐1,3‐benzodioxole HCl (MKC‐242) increased the release of DA and NA and decreased the release of 5‐HT in the frontal cortex of mice. The effect of MKC‐242 on DA release was significantly less in isolation‐reared mice than in group‐reared mice, while effects of the drug on NA and 5‐HT release did not differ between both groups. The effect of the other 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin on cortical DA release was also less in isolation‐reared mice than in group‐reared mice, and that of the drug on cortical 5‐HT release did not differ between both groups. In contrast to MKC‐242‐induced DA release, amphetamine‐induced increase in cortical DA release in vivo was greater in isolation‐reared mice. The present findings suggest that isolation rearing enhances the activity of cortical dopaminergic neurons and reduces selectively the 5‐HT1A receptor‐mediated release of DA in the cortex.
Stress‐mediated signaling in PC12 cells – the role of the small heat shock protein, Hsp27, and Akt in protecting cells from heat stress and nerve growth factor withdrawal
Authors: Mearow K. M. , Dodge M. E. , Rahimtula M. , Yegappan C.
Published: March 2003

We have investigated the role of stress‐activated signaling pathways and the small heat shock protein, Hsp27, in protecting PC12 cells from heat shock and nerve growth factor (NGF) withdrawal‐induced apoptosis. PC12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to heat shock. This resulted in the rapid activation of Akt followed by p38 mitogen‐activated protein kinase (MAPK) signaling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 was found to form an immunoprecipitable complex with Akt and p38 MAPK in both non‐stimulated and heat shocked cells, although after heat shock there was a gradual dissociation of Akt and p38 from the Hsp27. Cells were differentiated with NGF and then subjected to NGF withdrawal, a treatment which results in substantial cell death over 24–72 h. Hsp27 was shown to be protective against this treatment, since HSPC cells which overexpress Hsp27 showed significantly less cell death than the parental PC12 cells. In addition, we observed that phosphorylation of Akt was maintained in HSPC cells subjected to heat shock and NGF withdrawal compared with the parental cells. Taken together, our results suggest that Hsp27 may protect Akt from dephosphorylation and may also act in stabilizing Akt.
ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53
Authors: Bockmann J. , Kreutz M. R. , Gundelfinger E. D. , Böckers T. M.
Published: March 2003

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA‐type and metabotropic glutamate receptors and the actin‐based cytoskeleton. In a yeast two hybrid screen utilizing a proline‐rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co‐immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C‐terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline‐rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.
Regulation of taurine transport at the blood–brain barrier by tumor necrosis factor‐α, taurine and hypertonicity
Authors: Kang Young‐Sook , Ohtsuki Sumio , Takanaga Hitomi , Tomi Masatoshi , Hosoya Ken‐ichi , Terasaki Tetsuya
Published: March 2003

Taurine is the abundant sulfur‐containing β‐amino acid in brain where it exerts a neuroprotective effect. Although it is known that the blood–brain barrier (BBB) mediates taurine transport, the regulation of taurine transport have not been clarified yet. A conditionally immortalized rat brain capillary endothelial cells (TR‐BBB13), an in vitro model of the BBB, exhibited [3H]taurine uptake, which was dependent on both Na+ and Cl–, and inhibited by β‐alanine. Taurine transporter (TAUT) mRNA was detected in TR‐BBB13 cells, and TAUT protein was also expressed at 70 kDa. TR‐BBB13 cells exposed to 20 ng/mL TNF‐α and under hypertonic conditions showed a 1.7‐fold and 3.2‐fold increase in [3H]taurine uptake, respectively. In contrast, lipopolysaccharide and diethyl maleate did not significantly affect taurine uptake. The taurine uptake was reduced by pre‐treatment with excess taurine (50 mm). The mRNA level of the TAUT in TNF‐α and following hypertonic treatment was greater than that in control cells, whereas that under excess taurine conditions was lower than in controls. Therefore, taurine transport activity at the BBB appears to be regulated at the transcriptional level by cell damage, osmolality and taurine in the brain.
Oxidative modulation of nuclear factor‐κB in human cells expressing mutant fALS‐typical superoxide dismutases
Authors: Casciati Arianna , Ferri Alberto , Cozzolino Mauro , Celsi Fulvio , Nencini Monica , Rotilio Giuseppe , Carrì Maria Teresa
Published: March 2003

Previous evidence supports the notion of a redox regulation of protein phosphatase calcineurin that might be relevant for neurodegenerative processes where an imbalance between generation and removal of reactive oxygen species occurs. We have recently observed that calcineurin activity is depressed in human neuroblastoma cells expressing Cu,Zn superoxide dismutase (SOD1) mutant G93A and in brain areas from G93A transgenic mice, and that mutant G93A‐SOD1 oxidatively inactivates calcineurin in vitro. We have studied the possibility that, by interfering directly with calcineurin activity, mutant SOD1 can modulate pathways of signal transduction mediated by redox‐sensitive transcription factors. In this paper, we report a calcineurin‐dependent activation of nuclear factor‐κB (NF‐κB) induced by the expression of familial amyotrophic lateral sclerosis (fALS)‐SOD1s in human neuroblastoma cell lines. Alteration of the phosphorylation state of IκBα (the inhibitor of NF‐κB translocation into the nucleus) and induction of cyclooxygenase 2 are consistent with the up‐regulation of this transcription factor in this system. All of these modifications might be relevant to signaling pathways involved in the pathogenesis of fALS.
Manganese ethylene‐bis‐dithiocarbamate and selective dopaminergic neurodegeneration in rat: a link through mitochondrial dysfunction
Authors: Zhang Jing , Fitsanakis Vanessa A. , Gu Guangyu , Jing Deqiang , Ao Mingfang , Amarnath Venkataraman , Montine Thomas J.
Published: March 2003

Manganese ethylene‐bis‐dithiocarbamate (Mn‐EBDC) is the major active element of maneb, a pesticide linked to parkinsonism in certain individuals upon chronic exposure. Additionally, it has been shown to produce dopaminergic neurodegeneration in mice systemically coexposed to another pesticide, 1,1′‐dimethyl‐4,4′‐bipyridinium (paraquat). Here, we described a rat model in which selective dopaminergic neurodegeneration was produced by delivering Mn‐EBDC directly to the lateral ventricles. After establishing this model, we tested whether Mn‐EBDC provoked dopamine efflux in the striatum, a well‐known phenomenon produced by the mitochondrial inhibitor 1‐methyl‐4‐phenylpyridinium (MPP+), the active metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) that causes parkinsonism in humans, as well as in some animals. Finally, we investigated whether Mn‐EBDC directly inhibited mitochondrial function in vitro using isolated brain mitochondria. Our data demonstrated that Mn‐EBDC induced extensive striatal dopamine efflux that was comparable with that induced by MPP+, and that Mn‐EBDC preferentially inhibited mitochondrial complex III. As mitochondrial dysfunction is pivotal in the pathogenesis of Parkinson's disease (PD), our results support the proposal that exposure to pesticides such as maneb, or other naturally occurring compounds that inhibit mitochondrial function, may contribute to PD development.
Rhythmic changes in metabolism of dopamine in the chick retina: the importance of light versus biological clock
Authors: Zawilska Jolanta B. , Bednarek Aneta , Berezińska Małgorzata , Nowak Jerzy Z.
Published: March 2003

Rhythmic changes in dopamine (DA) content and metabolism were studied in retinas of chicks that were adapted to three different lighting conditions: 12‐h light : 12‐h dark (LD), constant darkness (DD) and continuous light (LL). Retinas of chicks kept under LD conditions exhibited light–dark‐dependent variations in the steady‐state level of DA and the two metabolites of DA, i.e. 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA). Concentrations of DA, DOPAC and HVA were high in light hours and low in dark hours of the LD illumination cycle. In retinas of chicks kept under DD, the content of DA, DOPAC and HVA oscillated in a rhythmic manner for 2 days, with higher values during the subjective light phase than during the subjective dark phase. The amplitudes of the observed oscillations markedly and progressively declined compared with the amplitudes recorded under the LD cycle. In retinas of chicks kept under LL conditions, levels of DA, DOPAC and HVA were similar to those found during the light phase of the LD cycle. Changes in the retinal contents of DA and HVA did not exhibit pronounced daily oscillations, while on the first day of LL the retinal concentrations of DOPAC were significantly higher during the subjective light phase than during the subjective dark phase. Acute exposure of chicks to light during the dark phase of the LD cycle markedly increased DA and DOPAC content in the retina. In contrast, light deprivation during the day decreased the retinal concentrations of DA and DOPAC. It is suggested that of the two regulatory factors controlling the level and metabolism of DA in the retina of chick, i.e. light and biological clock, environmental lighting conditions seem to be of major importance, with light conveying a stimulatory signal for the retinal dopaminergic cells.
Corticotropin‐releasing factor (CRF) rapidly suppresses apoptosis by acting upstream of the activation of caspases
Authors: Radulovic Marko , Hippel Cathrin , Spiess Joachim
Published: March 2003

The physiological role of the corticotropin‐releasing factor (CRF) family of peptides has recently been extended by emerging evidence of their cytoprotective effects. To determine whether CRF‐mediated cytoprotection is linked to caspase‐dependent apoptosis, the effect of CRF on the activation of caspases was investigated in detail in Y79 human retinoblastoma cells. The results presented here demonstrate that the cytoprotective effect of CRF against the actions of camptothecin (CT) was mediated by CRF receptor subtype 1, but not subtype 2. The observed CRF‐mediated cytoprotection involved rapid and pronounced suppression of proteolytic processing and activation of procaspase‐3, exerted even when CRF was added hours after the application of the cytotoxic agent. Surprisingly, activation of procaspase‐3 preceded activation of the initiator procaspases 2, 8, 9 and 10 during CT‐induced apoptosis of Y79 cells. The mechanism of the effect of CRF was examined using inhibitors of signalling pathways such as Wortmannin (Akt), cyclic AMP‐dependent protein kinase (PKA), extracellular signal‐regulated kinase (ERK), protein kinase c (PKC), p38 mitogen‐activated protein kinase (p38 MAPK), phospholipase c (PLC), nuclear factor‐κB (NF‐κΒ) and c‐jun N‐terminal kinase (JNK). The involvement of PKA in the mediation of the anti‐apoptotic effect of CRF has been established. Taken together, these results demonstrate for the first time that the cytoprotective effect of CRF involved suppression of pro‐apoptotic pathways at a site upstream of activation of procaspase‐3.
The Tat antagonist neomycin B hexa‐arginine conjugate inhibits gp‐120‐induced death of human neuroblastoma cells
Authors: Catani Maria Valeria , Corasaniti Maria Tiziana , Ranalli Marco , Amantea Diana , Litovchick Alexander , Lapidot Aviva , Melino Gerry
Published: March 2003

Several patients with acquired immunodeficiency syndrome (AIDS) develop neurological complications, which are referred to as human immunodeficiency virus (HIV)‐associated dementia (HAD). The HIV‐1 coat glycoprotein gp‐120 has been proposed as the major etiologic agent for neuronal loss reported postmortem in the brain of AIDS patients. Chemokine receptors may play a role in gp‐120‐triggered neurotoxicity, both in vitro and in vivo, thus being an intriguing target for developing therapeutic strategies aimed to prevent or reduce neuronal damage occurring during HIV infection. We have previously shown that human CHP100 neuroblastoma cells express CXCR4 and CCR5 chemokine receptors and that interaction between gp‐120 and these receptors contributes to cytotoxicity elicited by the protein. Here, we examined the neuroprotective potential of neomycin B hexa‐arginine conjugate (NeoR), a recently synthesized compound with anti‐HIV activity. We found that gp‐120‐triggered death is significantly reduced by NeoR, and this protective effect seems related to the ability of NeoR to interact with CXCR4 receptors. The ability of NeoR to cross the blood–brain barrier, as demonstrated in mice by systemic administration of the fluorescein conjugate drug, makes this compound a powerful and attractive therapeutic agent.
Patterns in midwives’ and expectant/new parents’ ways of relating to each other in ante‐ and postnatal consultations
Authors: Olsson Pia , Jansson Lilian
Published: March 2003

Patterns in midwives’ and expectant/new parents’ ways of relating to each other in ante‐ and postnatal consultations

Patterns in midwives’ and expectant/new parents’ ways of relating to each other in ante‐ and postnatal midwifery consultations were described by means of qualitative content analysis of 58 video‐recorded consultations in a Swedish primary care setting. The midwives appeared to steer the consultations by adopting a basic pattern and occasionally including other patterns. Of the five patterns disclosed, three were revealed as basic and named ‘the respectful gardener and her developing plants’, ‘the propagandist teacher and her ignorant pupils’ and ‘the steering inspector and the representatives of the population at disposal’. The midwives related in individually oriented ways in the first pattern while the latter two indicated generalized ways of relating. The expectant/new mothers were more active in the two additional patterns ‘the mediating counsellor and the discreet seekers of guidance’ and ‘the personal women‐friends’. The expectant/new fathers mostly had a minor role, only appearingly actively involved within the ‘gardener’ pattern. Midwives need to reflect over their ways of relating as these influence how expectant/new parents deal with the information given and their satisfaction with the care.