Chronic hyperammonemia alters in opposite ways membrane expression of GluA1 and GluA2 AMPA receptor subunits in cerebellum. Molecular mechanisms involved

Hyperammonemia contributes to altered neurotransmission and cognition in patients with hepatic encephalopathy. Hyperammonemia in rats affects differently high- and low-affinity AMPA receptors (AMPARs) in cerebellum. We hypothesized that hyperammonemia would alter differently membrane expression of AMPARs GluA1 and GluA2 subunits by altering its phosphorylation. This work aims were: 1) assess if hyperammonemia alters GluA1 and GluA2 subunits membrane expression in cerebellum and 2) analyze the underlying mechanisms.Hyperammonemia reduces membrane expression of GluA2 and enhances membrane expression of GluA1 in vivo. We show that changes in GluA2 and GluA1 membrane expression in hyperammonemia would be due to enhanced NMDA receptors activation which reduces cGMP levels and phosphodiesterase 2 (PDE2) activity, resulting in increased cAMP levels. This leads to increased protein kinase A (PKA) activity which activates phospholipase C (PLC) and protein kinase C (PKC) thus increasing phosphorylation of GluA2 in Ser880, which reduces GluA2 membrane expression, and phosphorylation of GluA1 in Ser831, which increases GluA1 membrane expression. Blocking NMDA receptors or inhibiting PKA, PLC or PKC normalizes GluA2 and GluA1 phosphorylation and membrane expression in hyperammonemic rats.Altered GluA2 and GluA1 membrane expression would alter signal transduction which may contribute to cognitive and motor alterations in hyperammonemia and hepatic encephalopathy.     

Protease‐activated receptor‐1 modulates hippocampal memory formation and synaptic plasticity

Protease‐activated receptor‐1 (PAR1) is an unusual G‐protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1?/? mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1?/? mice have deficits in hippocampus‐dependent memory. We also show that while PAR1?/? mice have normal baseline synaptic transmission at Schaffer collateral‐CA1 synapses, they exhibit severe deficits in N‐methyl‐d ‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR‐mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR‐dependent processes subserving memory formation and synaptic plasticity.     

Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV)

Vanadate has been recognized as a specific and potent phosphatase inhibitor since its structure is similar to that of phosphate. In this study, we measured the inhibition of glutathione S-transferase-tagged protein tyrosine phosphatase 1B (GST-PTP1B) and alkaline phosphatase (ALP) by the insulin enhancing compounds, bis(maltolato)oxovanadium(IV) (BMOV). The results showed that the activity of GST-PTP1B was reversibly inhibited by solutions of BMOV with an IC₅₀ value of 0.86 ± 0.02 μM. Steady state kinetic studies showed that inhibition of GST-PTP1B by BMOV was of a mixed competitive and noncompetitive type. In addition, incubation of GST-PTP1B with BMOV showed a time-dependent biphasic inactivation of the protein. On the other hand, the inhibitory behavior of BMOV on ALP activity was reversible and competitive with an IC₅₀ value of 32.1 ± 0.6 μM. Incubation with BMOV did not show biphasic inactivation of ALP. The reversible inhibition of GST-PTP1B by BMOV is more potent than that of ALP, but solutions of BMOV inhibited both enzymes. This data support the suggestion that mechanisms for the inhibitory effects of BMOV on GST-PTP1B and ALP are very different.     

Serotonin 5-HT receptor blockade enhances Ca(2+)/calmodulin-dependent protein kinase II function and membrane expression of AMPA receptor subunits in the rat hippocampus: implications for memory formation

Stimulation of hippocampal 5-HT(1A) receptors impairs memory retention. The highly selective 5-HT(1A) antagonist, WAY-100635, prevents the cognitive deficits induced not only by 5-HT(1A) stimulation but also by cholinergic or NMDA receptor blockade. On this basis, the effects of WAY-100635 on molecular events associated with memory storage were explored. In rat hippocampus, WAY-100635 produced a rapid increase in phosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and in Ca(2+)-independent CaMKII and protein kinase A (PKA) enzyme activity. This increase was followed a few hours later by an enhanced membrane expression of AMPA receptor subunits, especially of the GluR1 subunit phosphorylated at the CaMKII site, pGluR1(Ser831). The same qualitative effects were found with the weaker 5-HT(1A) antagonist NAN-190. The effects of both antagonists were no longer apparent in rats with a previous 5-HT depletion induced by the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), suggesting that 5-HT(1A) receptor blockade removes the tonic inhibition of 5-HT through 5-HT(1A) receptor stimulation on excitatory hippocampal neurons, with the consequent increase in PKA activity. In addition, administration of WAY-100635 potentiated the learning-specific increase in the hippocampus of phospho-CaMKII, Ca(2+)-independent CaMKII activity, as well as the phosphorylation of either the CaMKII or the PKA site on the AMPA receptor GluR1 subunit. This study suggests that blockade of hippocampal 5-HT(1A) receptors favours molecular events critically involved in memory formation, and provides an in vivo molecular basis for the proposed utility of 5-HT(1A) receptor antagonists in the treatment of cognitive disorders.     

Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells

BackgroundProtoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR.MethodsWe have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses.ResultsPP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression.ConclusionWe hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1.General significanceThese results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Region-specific enhancement of basal extracellular and cocaine-evoked dopamine levels following constitutive deletion of the Serotonin(1B) receptor

The behavioral effects of cocaine are enhanced following constitutive deletion of the serotonin(1B) receptor. The neural substrates mediating the enhanced response to cocaine are unknown. The present studies determined whether basal dopamine dynamics or cocaine-evoked dopamine levels are altered in projection areas of mesostriatal or mesoaccumbens dopamine neurons following serotonin(1B) receptor deletion. Male wild-type and serotonin(1B) knockout mice were implanted with microdialysis guide cannulas aimed at the dorsal striatum or nucleus accumbens. The zero net flux method of quantitative microdialysis was used to quantify basal extracellular dopamine concentrations (DA(ext)) and the extraction fraction of dopamine (E(d)), which provides an index of dopamine uptake. Conventional microdialysis techniques were used to quantify cocaine (0, 5.0, and 20.0 mg/kg)-evoked dopamine overflow. Basal DA(ext) and E(d) did not differ in striatum of wild-type and knockout mice. Similarly, cocaine-stimulated dopamine overflow did not differ between genotype. The basal E(d) did not differ in the nucleus accumbens of wild-type and knockout mice. However, DA(ext) was significantly elevated in the nucleus accumbens of knockout mice. Cocaine-evoked dopamine overflow (nM) was also enhanced in the nucleus accumbens of knockout mice. However, the cocaine-induced increase in dopamine levels, relative to basal values, did not differ between genotype. These data demonstrate that deletion of the serotonin(1B) receptor is associated with increases in basal DA(ext) in the nucleus accumbens. This increase is not associated with an alteration in E(d), suggesting increased basal dopamine release in these animals. It is hypothesized that these alterations in presynaptic neuronal activity are a compensatory response to constitutive deletion of the serotonin(1B) receptor and may contribute to the enhanced behavioral effects of psychostimulants observed in knockout mice.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

传染性喉气管炎病毒gB基因和新城疫病毒F基因在重组禽痘病毒中共表达

在重组禽痘病毒中表达多个禽类病原的主要免疫原基因是构建多价基因工程疫苗的前提,但相关研究很少。在表达传染性喉气管炎病毒(ILTV)gB基因重组禽痘病毒的转移载体的基础上,构建了含有ILTV gB基因和新城疫病毒(NDV)F基因的重组禽痘病毒转移载体pSY-gB-F,采用脂质体转染禽痘病毒感染的鸡胚成纤维(CEF)细胞后,通过蓝斑试验筛选出重组禽痘病毒(rFPv-gB-F),并进行了6轮蚀斑纯化。Western-blot试验和间接免疫荧光试验证明ILTV gB基因和NBVF基因在rFPV-gB-F感染的CEF细胞中获得表达。为传染性喉气管炎、新城疫与鸡痘活载体多价疫苗的研制奠定基础。     

牦牛B细胞淋巴瘤2相关蛋白A1的原核表达及体外活性

为研究牦牛(Bos grunniens) B细胞淋巴瘤2相关蛋白A1(B cell lymphoma 2 related protein A1,BCL2A1)的原核表达及体外活性。采用原核表达载体构建、细胞划痕实验、CCK-8法、透射电镜和实时荧光定量PCR等方法。结果显示,成功构建pET-32a-BCL2A1重组质粒,表达获得约33kDa的BCL2A1。终浓度为0.02μg/mL、0.2μg/mL、2.0μg/mL的BCL2A1均能使HepG2细胞活性显著降低(P < 0.05),并在一定程度上抑制细胞的迁移。2.0μg/mL BCL2A1导致HepG2细胞核固缩,胞质中高密度物质聚集,溶酶体吞噬形成致密的凋亡小体等。此外,细胞凋亡相关基因CASP9的mRNA水平在2.0 μg/mL组中显著上调(P < 0.05),CASP8的mRNA水平在0.2 μg/mL和2.0 μg/mL组中显著上调(P < 0.05),而CASP3和Cyt c的mRNA水平在3个浓度处理组均显著上调(P < 0.05)。这提示BCL2A1可能通过凋亡途径影响HepG2细胞活性,为进一步探索牦牛BCL2A1基因功能积累资料。     

Substance P N-terminal fragment SP(1-7) attenuates chronic morphine tolerance and affects dynorphin B and nociceptin in rats

The N-terminal substance P fragment SP_1-7 is known to modulate hyperalgesia and opioid withdrawal in animal models. This study examined the effects of intraperitoneal (i.p.) injections of SP_1-7 on chronic morphine tolerance and on the levels of dynorphin B (DYN B) and nociceptin/orphanin FQ (N/OFQ) in various brain areas of male Sprague-Dawley rats. Morphine tolerance was induced by subcutaneous injections of the opioid (10 mg/kg) twice daily for 7 days. SP_1-7 injected i.p. (185 nmol/kg) 30 min prior to morphine reduced the development of morphine tolerance. Immunoreactive (ir) DYN B and N/OFQ peptide levels were measured in several areas of the central nervous system. Levels of ir DYN B in rats treated with SP_1-7 and morphine were decreased in the nucleus accumbens, substantia nigra and ventral tegmental area and increased in the frontal cortex. The ir N/OFQ levels were increased in the periaqueductal gray and decreased in the nucleus accumbens. Since the concentration profiles of the two peptides were altered by SP_1-7 in the areas that are implicated in the modulation of opioid tolerance and analgesia, it is suggested that DYN B and N/OFQ systems may be involved in the effects of SP_1-7 on opioid tolerance.     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) in human placenta of the first, second, and third trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.     

High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice

Recent findings suggest that hypercholesterolemia may contribute to the onset of Alzheimer's disease-like dementia but the underlying mechanisms remain unknown. In this study, we evaluated the cognitive performance in rodent models of hypercholesterolemia in relation to neuroinflammatory changes and amyloid precursor protein (APP) processing, the two key parameters of Alzheimer's disease pathogenesis. Groups of normal C57BL/6 and low density lipoprotein receptor (LDLR)-deficient mice were fed a high fat/cholesterol diet for an 8-week period and tested for memory in a radial arm maze. It was found that the C57BL/6 mice receiving a high fat diet were deficient in handling an increasing working memory load compared with counterparts receiving a control diet while the hypercholesterolemic LDLR−/− mice showed impaired working memory regardless of diet. Immunohistochemical analysis revealed the presence of activated microglia and astrocytes in the hippocampi from high fat-fed C57BL/6 mice and LDLR−/− mice. Consistent with a neuroinflammatory response, the hyperlipidemic mice showed increased expression of cytokines/mediators including tumor necrosis factor-α, interleukin-1β and -6, nitric oxide synthase 2, and cycloxygenase 2. There was also an induced expression of the key APP processing enzyme i.e. β-site APP cleaving enzyme 1 in both high fat/cholesterol-fed C57BL/6 and LDLR−/− mice accompanied by an increased generation of C-terminal fragments of APP. Although ELISA for beta-amyloid failed to record significant changes in the non-transgenic mice, a threefold increase in beta-amyloid 40 accumulation was apparent in a strain of transgenic mice expressing wild-type human APP on high fat/cholesterol diet. The findings link hypercholesterolemia with cognitive dysfunction potentially mediated by increased neuroinflammation and APP processing in a non-transgenic mouse model.     

Regulation of dopamine D-receptor activation in vivo by protein phosphatase 2B (calcineurin)

Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1-agonist-stimulated [35S]GTPgammaS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPgammaS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal and D1-agonist-stimulated phosphorylation of D1-receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increased kinase-mediated phosphorylation.     

Activation of 5-HT(1A) but not 5-HT(1B) receptors attenuates an increase in extracellular dopamine derived from exogenously administered L-DOPA in the striatum with nigrostriatal denervation

In order to determine whether L-DOPA-derived extracellular dopamine (DA) in the striatum with dopaminergic denervation is affected by activation of serotonin autoreceptors (5-HT(1A) and 5-HT(1B) receptors), we applied in vivo brain microdialysis technique to 6-hydroxydopamine-lesioned rats and examined the effects of the selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the selective 5-HT(1B) receptor agonist CGS-12066 A on L-DOPA-derived extracellular DA levels. Single L-DOPA injection (50 mg/kg i.p.) caused a rapid increase and a following decrease of extracellular DA, with a peak value at 100 min after L-DOPA injection. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8-OH-DPAT (i.p.) significantly attenuated an increase in L-DOPA-derived extracellular DA and the times of peak DA levels were prolonged to 150 min and 225 min after L-DOPA injection, respectively. These 8-OH-DPAT-induced changes in L-DOPA-derived extracellular DA were antagonized by further pretreatment with WAY-100635, a selective 5-HT(1A) antagonist. In contrast, intrastriatal perfusion with the 5-HT(1B) agonist CGS-12066 A (10 nM and 100 nM) did not induce any changes in L-DOPA-derived extracellular DA. Thus, stimulation of 5-HT(1A) but not 5-HT(1B) receptors attenuated an increase in extracellular DA derived from exogenous L-DOPA. These results support the hypothesis that serotonergic neurons are primarily responsible for the storage and release of DA derived from exogenous L-DOPA in the absence of dopaminergic neurons.     

Sustained activation of metabotropic glutamate receptor 5 and protein tyrosine phosphatases mediate the expression of (S)-3,5-dihydroxyphenylglycine-induced long-term depression in the hippocampal CA1 region

Previous studies have shown that brief application of group I metabotropic glutamate receptor (mGluR) agonist (S)-3, 5-dihydroxyphenylglycine (DHPG) to hippocampal slices can induce a chemical form of long-term depression (DHPG-LTD) in the hippocampal CA1 region; however, the expression mechanisms of this LTD remain unclear. We show here that the expression of DHPG-LTD can be specifically reversed by application of the broad-spectrum mGluR antagonists, (S)-alpha-methyl-4-carboxyphenylglycine (MCPG) and LY341495, and mGluR5 antagonist, 2-methyl-6-(phenylethyl)pyridine, but not by NMDA receptor antagonist, D-2-amino-5-phosphonopentanoic acid, mGluR1 antagonist, LY367385, group II mGluR antagonist, (2S)-alpha-ethylglutamic acid, or group III mGluR antagonist, (S)-2-amino-2-methyl-4-phosphonobutanic acid (MAP4). In addition, the ability of MCPG to reverse DHPG-LTD was mimicked by the protein tyrosine phosphatase inhibitors, phenylarsine oxide and orthovanadate, but not phospholipase C inhibitor, U73122, protein kinase C inhibitor, bisindolylmaleimide 1, p38 mitogen-activated protein kinase inhibitor, SB203580, or protein phosphatases 1/2 A inhibitor, okadaic acid. Moreover, MCPG reversed the DHPG-LTD without affecting the paired-pulse facilitation. The expression of DHPG-LTD was associated with the reduction of both tyrosine phosphorylation and surface expression of AMPA receptor GluR2 subunits. Together, these results suggest that sustained activation of mGluR5 and in turn triggering a protein tyrosine phosphatase-dependent regulation of postsynaptic expression of AMPA receptors may contribute to the expression of DHPG-LTD.     

Hippocampal gene expression profiling reveals the possible involvement of Homer1 and GABA(B) receptors in scopolamine-induced amnesia

Scopolamine-treated rats are commonly used as a psychopharmacological model of memory dysfunction and have been extensively studied to establish the effectiveness of acetylcholinesterase inhibitors in the treatment of Alzheimer's disease. Scopolamine is a muscarinic acetylcholine receptor antagonist that induces memory deficits in young subjects similar to those occurring during aging. The amnesic effect of scopolamine is well established but the molecular and cellular mechanisms that sustain its neuropharmacological action are still unclear. The present genome wide study investigates hippocampal gene expression profiling in scopolamine-treated adult rats following stimulation in a spatial memory task. Using microarray and quantitative real-time RT-PCR approaches, we identified several genes previously known to be associated with memory processes (Homer1, GABA(B) receptor, early growth response 1, prodynorphin, VGF nerve growth factor inducible) and multiple novel candidate genes possibly involved in cognition (including calcium/calmodulin-dependent protein kinase kinase 2, dual specificity phosphatase 5 and 6, glycophorin C) that were altered following scopolamine treatment. Moreover, we found that stable over-expression of glutamatergic components Homer1a and 1c in the hippocampus of adult rats induced by recombinant adeno-associated virus vector abolished memory improvement produced by the GABA(B) receptor antagonist SGS742 in scopolamine-treated rats. Taken together, these results reveal novel genes and mechanisms involved in scopolamine-induced amnesia, and demonstrate the involvement of both GABA and glutamate neurotransmission in this animal model of cognitive dysfunctions.     

Regional changes in density of serotonin transporter in the brain of 5-HT1A and 5-HT1B knockout mice, and of serotonin innervation in the 5-HT1B knockout

5-HT1A knockout (KO) mice display an anxious-like phenotype, whereas 5-HT1B KOs are over-aggressive. To identify serotoninergic correlates of these altered behaviors, autoradiographic measurements of 5-HT1A and 5-HT1B serotonin (5-HT) receptors and transporter (5-HTT) were obtained using the radioligands [3H]8-OH-DPAT, [125I]cyanopindolol and [3H]citalopram, respectively. By comparison to wild-type, density of 5-HT1B receptors was unchanged throughout brain in 5-HT1A KOs, and that of 5-HT1A receptors in 5-HT1B KOs. In contrast, decreases in density of 5-HTT binding were measured in several brain regions of both genotypes. Moreover, 5-HTT binding density was significantly increased in the amygdalo-hippocampal nucleus and ventral hippocampus of the 5-HT1B KOs. Measurements of 5-HT axon length and number of axon varicosities by quantitative 5-HT immunocytochemistry revealed proportional increases in the density of 5-HT innervation in these two regions of 5-HT1B KOs, whereas none of the decreases in 5-HTT binding sites were associated with any such changes. Several conclusions could be drawn from these results: (i) 5-HT1B receptors do not adapt in 5-HT1A KOs, nor do 5-HT1A receptors in 5-HT1B KOs. (ii) 5-HTT is down-regulated in several brain regions of 5-HT1A and 5-HT1B KO mice. (iii) This down-regulation could contribute to the anxious-like phenotype of the 5-HT1A KOs, by reducing 5-HT clearance in several territories of 5-HT innervation. (iv) The 5-HT hyperinnervation in the amygdalo-hippocampal nucleus and ventral hippocampus of 5-HT1B KOs could play a role in their increased aggressiveness, and might also explain their better performance in some cognitive tests. (v) These increases in density of 5-HT innervation provide the first evidence for a negative control of 5-HT neuron growth mediated by 5-HT1B receptors.