Sample records for ampa receptor subunit

  1. Differences in AMPA and Kainate Receptor Interactomes Facilitate Identification of AMPA Receptor Auxiliary Subunit GSG1L

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    Natalie F. Shanks


    Full Text Available AMPA receptor (AMPA-R complexes consist of channel-forming subunits, GluA1-4, and auxiliary proteins, including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. The combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors, we compared the proteins that copurify with each receptor type in the rat brain. This interactome study identified the majority of known interacting proteins and, more importantly, provides candidates for additional studies. We validate the claudin homolog GSG1L as a newly identified binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes, and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function.

  2. Auxiliary Subunits: Shepherding AMPA Receptors to the Plasma Membrane

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    Simon C. Haering


    Full Text Available Ionotropic glutamate receptors (iGluRs are tetrameric ligand-gated cation channels that mediate excitatory signal transmission in the central nervous system (CNS of vertebrates. The members of the iGluR subfamily of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors (AMPARs mediate most of the fast excitatory signal transmission, and their abundance in the postsynaptic membrane is a major determinant of the strength of excitatory synapses. Therefore, regulation of AMPAR trafficking to the postsynaptic membrane is an important constituent of mechanisms involved in learning and memory formation, such as long-term potentiation (LTP and long-term depression (LTD. Auxiliary subunits play a critical role in the facilitation and regulation of AMPAR trafficking and function. The currently identified auxiliary subunits of AMPARs are transmembrane AMPA receptor regulatory proteins (TARPs, suppressor of lurcher (SOL, cornichon homologues (CNIHs, synapse differentiation-induced gene I (SynDIG I, cysteine-knot AMPAR modulating proteins 44 (CKAMP44, and germ cell-specific gene 1-like (GSG1L protein. In this review we summarize our current knowledge of the modulatory influence exerted by these important but still underappreciated proteins.

  3. Effects of visual deprivation during brain development on expression of AMPA receptor subunits in rat’s hippocampus

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    Sayyed Alireza Talaei


    Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

  4. C-terminal interactors of the AMPA receptor auxiliary subunit Shisa9.

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    Anna R Karataeva

    Full Text Available Shisa9 (initially named CKAMP44 has been identified as auxiliary subunit of the AMPA-type glutamate receptors and was shown to modulate its physiological properties. Shisa9 is a type-I transmembrane protein and contains a C-terminal PDZ domain that potentially interacts with cytosolic proteins. In this study, we performed a yeast two-hybrid screening that yielded eight PDZ domain-containing interactors of Shisa9, which were independently validated. The identified interactors are known scaffolding proteins residing in the neuronal postsynaptic density. To test whether C-terminal scaffolding interactions of Shisa9 affect synaptic AMPA receptor function in the hippocampus, we disrupted these interactions using a Shisa9 C-terminal mimetic peptide. In the absence of scaffolding interactions of Shisa9, glutamatergic AMPA receptor-mediated synaptic currents in the lateral perforant path of the mouse hippocampus had a faster decay time, and paired-pulse facilitation was reduced. Furthermore, disruption of the PDZ interactions between Shisa9 and its binding partners affected hippocampal network activity. Taken together, our data identifies novel interaction partners of Shisa9, and shows that the C-terminal interactions of Shisa9 through its PDZ domain interaction motif are important for AMPA receptor synaptic and network functions.

  5. Individual stress vulnerability is predicted by short-term memory and AMPA receptor subunit ratio in the hippocampus. (United States)

    Schmidt, Mathias V; Trümbach, Dietrich; Weber, Peter; Wagner, Klaus; Scharf, Sebastian H; Liebl, Claudia; Datson, Nicole; Namendorf, Christian; Gerlach, Tamara; Kühne, Claudia; Uhr, Manfred; Deussing, Jan M; Wurst, Wolfgang; Binder, Elisabeth B; Holsboer, Florian; Müller, Marianne B


    Increased vulnerability to aversive experiences is one of the main risk factors for stress-related psychiatric disorders as major depression. However, the molecular bases of vulnerability, on the one hand, and stress resilience, on the other hand, are still not understood. Increasing clinical and preclinical evidence suggests a central involvement of the glutamatergic system in the pathogenesis of major depression. Using a mouse paradigm, modeling increased stress vulnerability and depression-like symptoms in a genetically diverse outbred strain, and we tested the hypothesis that differences in AMPA receptor function may be linked to individual variations in stress vulnerability. Vulnerable and resilient animals differed significantly in their dorsal hippocampal AMPA receptor expression and AMPA receptor binding. Treatment with an AMPA receptor potentiator during the stress exposure prevented the lasting effects of chronic social stress exposure on physiological, neuroendocrine, and behavioral parameters. In addition, spatial short-term memory, an AMPA receptor-dependent behavior, was found to be predictive of individual stress vulnerability and response to AMPA potentiator treatment. Finally, we provide evidence that genetic variations in the AMPA receptor subunit GluR1 are linked to the vulnerable phenotype. Therefore, we propose genetic variations in the AMPA receptor system to shape individual stress vulnerability. Those individual differences can be predicted by the assessment of short-term memory, thereby opening up the possibility for a specific treatment by enhancing AMPA receptor function.

  6. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons (United States)

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.


    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  7. The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons (United States)

    Inglis, Fiona M.; Crockett, Richard; Korada, Sailaja; Abraham, Wickliffe C.; Hollmann, Michael; Kalb, Robert G.


    The morphology of the mature motor neuron dendritic arbor is determined by activity-dependent processes occurring during a critical period in early postnatal life. The abundance of the AMPA receptor subunit GluR1 in motor neurons is very high during this period and subsequently falls to a negligible level. To test the role of GluR1 in dendrite morphogenesis, we reintroduced GluR1 into rat motor neurons at the end of the critical period and quantitatively studied the effects on dendrite architecture. Two versions of GluR1 were studied that differed by the amino acid in the "Q/R" editing site. The amino acid occupying this site determines single-channel conductance, ionic permeability, and other essential electrophysiologic properties of the resulting receptor channels. We found large-scale remodeling of dendritic architectures in a manner depending on the amino acid occupying the Q/R editing site. Alterations in the distribution of dendritic arbor were not prevented by blocking NMDA receptors. These observations suggest that the expression of GluR1 in motor neurons modulates a component of the molecular substrate of activity-dependent dendrite morphogenesis. The control of these events relies on subunit-specific properties of AMPA receptors.

  8. Auxiliary Subunit GSG1L Acts to Suppress Calcium-Permeable AMPA Receptor Function (United States)

    McGee, Thomas P.; Bats, Cécile


    AMPA-type glutamate receptors are ligand-gated cation channels responsible for a majority of the fast excitatory synaptic transmission in the brain. Their behavior and calcium permeability depends critically on their subunit composition and the identity of associated auxiliary proteins. Calcium-permeable AMPA receptors (CP-AMPARs) contribute to various forms of synaptic plasticity, and their dysfunction underlies a number of serious neurological conditions. For CP-AMPARs, the prototypical transmembrane AMPAR regulatory protein stargazin, which acts as an auxiliary subunit, enhances receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines. We find that, in contrast, GSG1L, a transmembrane auxiliary protein identified recently as being part of the AMPAR proteome, acts to reduce the weighted mean single-channel conductance and calcium permeability of recombinant CP-AMPARs, while increasing polyamine-dependent rectification. To examine the effects of GSG1L on native AMPARs, we manipulated its expression in cerebellar and hippocampal neurons. Transfection of GSG1L into mouse cultured cerebellar stellate cells that lack this protein increased the inward rectification of mEPSCs. Conversely, shRNA-mediated knockdown of endogenous GSG1L in rat cultured hippocampal pyramidal neurons led to an increase in mEPSC amplitude and in the underlying weighted mean single-channel conductance, revealing that GSG1L acts to suppress current flow through native CP-AMPARs. Thus, our data suggest that GSG1L extends the functional repertoire of AMPAR auxiliary subunits, which can act not only to enhance but also diminish current flow through their associated AMPARs. SIGNIFICANCE STATEMENT Calcium-permeable AMPA receptors (CP-AMPARs) are an important group of receptors for the neurotransmitter glutamate. These receptors contribute to various forms of

  9. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes. (United States)

    Dawe, G Brent; Musgaard, Maria; Aurousseau, Mark R P; Nayeem, Naushaba; Green, Tim; Biggin, Philip C; Bowie, Derek


    Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits.

  10. Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse. (United States)

    Panicker, Sandip; Brown, Keith; Nicoll, Roger A


    Glutamate is the primary excitatory neurotransmitter in the brain, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1-4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2(-/-) knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.

  11. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain

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    Amanda Lorraine Wright


    Full Text Available AMPA receptors are comprised of different combinations of GluR1-GluR4 (also known as GluA1-GluA4 and GluR-A to GluR-D subunits. The GluR2 subunit is subject to Q/R site RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Q, present in the GluR2 gene, to a codon for arginine (R found in the mRNA. AMPA receptors are calcium (Ca2+-permeable if they contain the unedited GluR2(Q subunit or if they lack the GluR2 subunit. While most AMPA receptors in the brain contain the edited GluR2(R subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable GluR2-lacking AMPA receptors are important in synaptic plasticity and learning. However, the presence of Ca2+-permeable AMPA receptors containing unedited GluR2 leads to excitotoxic cell loss. Recent studies have indicated that RNA editing of GluR2 is deregulated in diseases, such as amyotrophic lateral sclerosis (ALS, as well in acute neurodegenerative conditions, such as ischemia. More recently, studies have investigated the regulation of RNA editing and possible causes for its deregulation during disease. In this review, we will explore the role of GluR2 RNA editing in the healthy and diseased brain and outline new insights into the mechanisms that control this process.

  12. Reinforcement-related regulation of AMPA glutamate receptor subunits in the ventral tegmental area enhances motivation for cocaine. (United States)

    Choi, Kwang Ho; Edwards, Scott; Graham, Danielle L; Larson, Erin B; Whisler, Kimberly N; Simmons, Diana; Friedman, Allyson K; Walsh, Jessica J; Rahman, Zia; Monteggia, Lisa M; Eisch, Amelia J; Neve, Rachael L; Nestler, Eric J; Han, Ming-Hu; Self, David W


    Chronic cocaine use produces numerous biological changes in brain, but relatively few are functionally associated with cocaine reinforcement. Here we show that daily intravenous cocaine self-administration, but not passive cocaine administration, induces dynamic upregulation of the AMPA glutamate receptor subunits GluR1 and GluR2 in the ventral tegmental area (VTA) of rats. Increases in GluR1 protein and GluR1(S845) phosphorylation are associated with increased GluR1 mRNA in self-administering animals, whereas increased GluR2 protein levels occurred despite substantial decreases in GluR2 mRNA. We investigated the functional significance of GluR1 upregulation in the VTA on cocaine self-administration using localized viral-mediated gene transfer. Overexpression of GluR1(WT) in rat VTA primarily infected dopamine neurons (75%) and increased AMPA receptor-mediated membrane rectification in these neurons with AMPA application. Similar GluR1(WT) overexpression potentiated locomotor responses to intra-VTA AMPA, but not NMDA, infusions. In cocaine self-administering animals, overexpression of GluR1(WT) in the VTA markedly increased the motivation for cocaine injections on a progressive ratio schedule of cocaine reinforcement. In contrast, overexpression of protein kinase A-resistant GluR1(S845A) in the VTA reduced peak rates of cocaine self-administration on a fixed ratio reinforcement schedule. Neither viral vector altered sucrose self-administration, and overexpression of GluR1(WT) or GluR1(S845A) in the adjacent substantia nigra had no effect on cocaine self-administration. Together, these results suggest that dynamic regulation of AMPA receptors in the VTA during cocaine self-administration contributes to cocaine addiction by acting to facilitate subsequent cocaine use.

  13. AMPA receptors in the rat and primate hippocampus: a possible absence of GluR2/3 subunits in most interneurons. (United States)

    Leranth, C; Szeidemann, Z; Hsu, M; Buzsáki, G


    Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are assembled from the four subunits GluR1, 2, 3, 4 (or GluRA, B, C, D). AMPA channels that do not contain the GluR2 subunit are permeable to calcium. Recent studies indicate that excitotoxic as well as epileptic and ischemic cell damage may be mediated not only by N-methyl-Daspartate receptors, but also by AMPA receptors. The majority of interneurons in the hippocampus are resistant, but subsets of interneurons are consistently damaged in different disease states. Single immunolabeling using antibodies against AMPA receptor subunits, together with double immunolabeling for calcium-binding proteins (parvalbumin, calbindin and calretinin) and the neuropeptide somatostatin, were performed to study GluR1-4 immunoreactivity in interneuronal populations and principal cells. The ultrastructure of GluR1-4 labeled neurons was also examined using electron microscopy. With the exception of calbindin-positive interneurons, GluR2/3 was absent from hippocampal interneurons in both rat and monkey. In the rat, interneurons were more strongly immunoreactive against GluR1 than principal cells. In the monkey, immunoreactivity for GluR4 in interneurons was stronger than for GluR1. All GluR subunits were confined to spines, dendritic membrane and cytoplasm surrounding the nucleus but absent from axons and presynaptic terminals. Our findings suggest that hippocampal principal cells and interneurons express different complements of AMPA receptor subunits. Furthermore, the absence of GluR2 and/or GluR3 in both vulnerable and resistant interneurons subtypes indicates that knowledge of receptor subunit composition is not sufficient to predict neuronal vulnerability.

  14. AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus. (United States)

    Catania, M V; Bellomo, M; Giuffrida, R; Giuffrida, R; Stella, A M; Albanese, V


    Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.

  15. Administration of a PTEN inhibitor BPV(pic) attenuates early brain injury via modulating AMPA receptor subunits after subarachnoid hemorrhage in rats. (United States)

    Chen, Yujie; Luo, Chunxia; Zhao, Mingyue; Li, Qiang; Hu, Rong; Zhang, John H; Liu, Zhi; Feng, Hua


    The aim of this study was to investigate whether the phosphatase and tensin homolog deleted on chromosome ten (PTEN) inhibitor dipotassium bisperoxo(pyridine-2-carboxyl) oxovanadate (BPV(pic)) attenuates early brain injury by modulating α-amino-3-hydroxy-5-methyl-4-isoxa-zolep-propionate (AMPA) receptor subunits after subarachnoid hemorrhage (SAH). A standard intravascular perforation model was used to produce the experimental SAH in Sprague-Dawley rats. BPV(pic) treatment (0.2mg/kg) was evaluated for effects on neurological score, brain water content, Evans blue extravasation, hippocampal neuronal death and AMPA receptor subunits alterations after SAH. We found that BPV(pic) is effective in attenuating BBB disruption, lowering edema, reducing hippocampal neural death and improving neurological outcomes. In addition, the AMPA receptor subunit GluR1 protein expression at cytomembrane was downregulated, whereas the expression of GluR2 and GluR3 was upregulated after BPV(pic) treatment. Our results suggest that PTEN inhibited by BPV(pic) plays a neuroprotective role in SAH pathophysiology, possibly by alterations in glutamate AMPA receptor subunits.

  16. Differential dendritic targeting of AMPA receptor subunit mRNAs in adult rat hippocampal principal neurons and interneurons. (United States)

    Cox, David J; Racca, Claudia


    In hippocampal neurons, AMPA receptors (AMPARs) mediate fast excitatory postsynaptic responses at glutamatergic synapses, and are involved in various forms of synaptic plasticity. Dendritic local protein synthesis of selected AMPAR subunit mRNAs is considered an additional mechanism to independently and rapidly control the strength of individual synapses. We have used fluorescent in situ hybridization and immunocytochemistry to analyze the localization of AMPAR subunit (GluA1-4) mRNAs and their relationship with the translation machinery in principal cells and interneurons of the adult rat hippocampus. The mRNAs encoding all four AMPAR subunits were detected in the somata and dendrites of CA3 and CA1 pyramidal cells and those of six classes of CA1 γ-aminobutyric acid (GABA)ergic interneurons. GluA1-4 subunit mRNAs were highly localized to the apical dendrites of pyramidal cells, whereas in interneurons they were present in multiple dendrites. In contrast, in the dentate gyrus, GluA1-4 subunit mRNAs were virtually restricted to the somata and were absent from the dendrites of granule cells. These different regional and cell type-specific labeling patterns also correlated with the localization of markers for components of the protein synthesis machinery. Our results support the local translation of GluA1-4 mRNAs in dendrites of hippocampal pyramidal cells and CA1 interneurons but not in granule cells of the dentate gyrus. Furthermore, the regional and cell type-specific differences we observed suggest that each cell type uses distinct ways of regulating the local translation of AMPAR subunits.

  17. AMPA receptor ligands

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    Strømgaard, Kristian; Mellor, Ian


    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR), subtype of the ionotropic glutamate receptors (IGRs), mediate fast synaptic transmission in the central nervous system (CNS), and are involved in many neurological disorders, as well as being a key player in the f...

  18. Increase of AMPA receptor glutamate receptor 1 subunit and B-cell receptor-associated protein 31 gene expression in hippocampus of fatigued mice. (United States)

    Kamakura, Masaki; Tamaki, Keisuke; Sakaki, Toshiyuki; Yoneda, Yukio


    Central fatigue is an indispensable biosignal for maintaining life, but the neuronal and molecular mechanisms involved remain unclear. In this study, we searched for genes differentially expressed in the hippocampus of fatigued mice to elucidate the mechanisms underlying fatigue. Mice were forced to swim in an adjustable-current water pool, and the maximum swimming time (endurance) until fatigue was measured thrice. Fatigued and nonfatigued mice with equal swimming capacity and body weight were compared. We found that the genes of GluR1 and B-cell receptor-associated protein 31 (Bap31), which acts as a transport molecule in the secretory pathway or as a mediator of apoptosis, were upregulated in the hippocampus of fatigued mice, and increases of GluR1 and Bap31 were confirmed by Northern blotting and real-time PCR. No change of gene expression of AMPA receptor subunits other than GluR1 was observed. These results suggest that a compositional change of AMPA receptor (increase of GluR1) and upregulation of the Bap31 gene may be implicated in fatigue in mice.

  19. Extinction of morphine-dependent conditioned behavior is associated with increased phosphorylation of the GluR1 subunit of AMPA receptors at hippocampal synapses


    Billa, Sophie K.; Sinha, Namita; Rudrabhatla, Sri Rajyalakshmi; Morón, Jose A.


    In abstinent opiate addicts, relapse can be triggered by exposure to environmental cues associated with drug use; thus, the disruption of these learned associations may be an effective approach for reducing relapse. Interestingly, glutamatergic systems are thought to be involved in opiate-induced behavioral plasticity. In this study, changes in expression and phosphorylation levels of AMPA glutamate receptor subunits (GluR1, GluR2) in the hippocampus were investigated in rats showing a condit...

  20. Uncompetitive antagonism of AMPA receptors

    DEFF Research Database (Denmark)

    Andersen, Trine F; Tikhonov, Denis B; Bølcho, Ulrik;


    Philanthotoxins are uncompetitive antagonists of Ca2+-permeable AMPA receptors presumed to bind to the pore-forming region, but a detailed molecular mechanism for this interaction is missing. Here a small library of novel philanthotoxins was designed and synthesized using a solid-phase strategy. ...... polyamine toxins antagonize the AMPA receptor ion channel and provide the basis for rational development of uncompetitive antagonists of AMPA receptors....

  1. High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus. (United States)

    Baude, A; Nusser, Z; Molnár, E; McIlhinney, R A; Somogyi, P


    The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All subunits were enriched in the neuropil of the dendritic layers of the hippocampus and in the molecular layer of the dentate gyrus. The cellular distribution of the GluRD subunit was studied more extensively. The strata radiatum, oriens and the dentate molecular layer were more strongly immunoreactive than the stratum lacunosum moleculare, the stratum lucidum and the hilus. However, in the stratum lucidum of the CA3 area and in the hilus the weakly reacting dendrites were surrounded by immunopositive rosettes, shown in subsequent electron microscopic studies to correspond to complex dendritic spines. In the stratum radiatum, the weakly reacting apical dendrites contrasted with the surrounding intensely stained neuropil. The cell bodies of pyramidal and granule cells were moderately reactive. Some non-principal cells and their dendrites in the pyramidal cell layer and in the alveus also reacted very strongly for the GluRD subunit. At the subcellular level, silver intensified immunogold

  2. Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

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    Zhe eJin


    Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

  3. Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model (United States)

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.


    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

  4. Age-dependent modifications of AMPA receptor subunit expression levels and related cognitive effects in 3xTg-AD mice

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    Pamela eCantanelli


    Full Text Available GluA1, GluA2, GluA3, and GluA4 are the constitutive subunits of AMPA receptors (AMPARs, the major mediators of fast excitatory transmission in the mammalian central nervous system. Most AMPARs are Ca2+-impermeable because of the presence of the GluA2 subunit. GluA2 mRNA undergoes an editing process that results in a Q to R substitution, a key factor in the regulation of AMPAR Ca2+-permeability. AMPARs lacking GluA2 or containing the unedited subunit are permeable to Ca2+ and Zn2+. The phenomenon physiologically modulates synaptic plasticity while, in pathologic conditions, leads to increased vulnerability to excitotoxic neuronal death. Given the importance of these subunits, we have therefore evaluated possible associations between changes in expression levels of AMPAR subunits and development of cognitive deficits in 3xTg-AD mice, a widely investigated transgenic mouse model of Alzheimer’s disease. With qRT-PCR, we assayed hippocampal mRNA expression levels of GluA1-4 subunits occurring in young [3 months of age (m.o.a.] and old (12 m.o.a Tg-AD mice and made comparisons with levels found in age-matched wild type (WT mice. Efficiency of GluA2 RNA editing was also analyzed. All animals were cognitively tested for short- and long-term spatial memory with the Morris Water Maze (MWM navigation task. 3xTg-AD mice showed age-dependent decreases of mRNA levels for all the AMPAR subunits, with the exception of GluA2. Editing remained fully efficient with aging in 3xTg-AD and WT mice. A one-to-one correlation analysis between MWM performances and GluA1-4 mRNA expression profiles showed negative correlations between GluA2 levels and MWM performances in young 3xTg-AD mice. On the contrary, positive correlations between GluA2 mRNA and MWM performances were found in young WT mice. Our data suggest that increases of AMPARs that contain GluA1, GluA3, and GluA4 subunits may help in maintaining cognition in pre-symptomatic 3xTg-AD mice.

  5. Effects of ketamine-midazolam anesthesia on the expression of NMDA and AMPA receptor subunit in the peri-infarction of rat brain

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yue-lin; ZHANG Peng-bo; QIU Shu-dong; LIU Yong; TIAN Ying-fang; WANG Ying


    Background Activation of N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors play an important role in the neurons death induced by ischemia.The mitigating effect of intravenous anesthetics on ischemic neuron injury is related to their influence on NMDA receptors. This study was performed to investigate the effect of ketamine-midazolam anesthesia on the NMDA and AMPA receptor subunits expression in the peri-infarction of ischemic rat brain and explore its potential mechanism of neuroprotection.This study was supported by National Natural Science Foundation of China (NSFC) (No.30200291).Methods Thirty Sprague Dawley (SD) rats were subjected to permanent middle cerebral artery occlusion under ketamine/atropine (100/0.05 mg/kg) or ketamine-midazolam/atropine (60/50/0.05 mg/kg) intraperitoneal anesthesia (n=15 each). Twenty-four hours after ischemia, five rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally and infarct size was measured. Twenty-four and 72 hours after ischemia, four rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally. After staining the brain tissue slices with toluidine blue, the survived neurons in the peri-infarction were observed. Also, the expression level of NMDA receptors 1 (NR1), NMDA receptors 2A (NR2A), NMDA receptors 2B (NR2B) and AMPA (GluR1 subunit) were determined by grayscale analysis in immunohistochemical stained slices.Results Compared with ketamine anesthesia, ketamine-midazolam anesthesia produced not only smaller infarct size [(24.1±4.6)% vs (38.4±4.2)%, P<0.05], but also higher neuron density (24 hours: 846± 16 vs 756±24,P<0.05; 72 hours: 882±22 vs 785± 18, P<0.05) and lower NR2A (24 hours: 123.0±4.9 vs 95.0±2.5, P<0.05; 72 hours: 77.8±4.1 vs 54.2±3.9, P<0.05) and NR2B (24 hours: 98.5±2.7 vs 76.3±2.4, P<0.05; 72hours: 67.2

  6. Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia (United States)

    Akbarian, S.; Smith, M. A.; Jones, E. G.; Bloom, F. E. (Principal Investigator)


    Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA

  7. Cortical kindling induces elevated levels of AMPA and GABA receptor subunit mRNA within the amygdala/piriform region and is associated with behavioral changes in the rat. (United States)

    Henderson, Amy K; Galic, Michael A; Teskey, G Campbell


    Cortical kindling causes alterations within the motor cortex and results in long-standing motor deficits. Less attention has been directed to other regions that also participate in the epileptiform activity. We examined if cortical kindling could induce changes in excitatory and inhibitory receptor subunit mRNA in the amygdala/piriform regions and if such changes are associated with behavioral deficits. After cortical kindling, amygdala/piriform regions were dissected to analyze mRNA levels of NMDA, AMPA, and GABA receptor subunits using reverse transcription polymerase chain reaction, or rats were subjected to a series of behavioral tests. Kindled rats had significantly greater amounts of GluR1 and GluR2 AMPA receptor mRNA, and alpha1 and alpha2 GABA receptor subunit mRNA, compared with sham controls, which was associated with greater anxiety-like behaviors in the elevated plus maze and reduced freezing behaviors in the fear conditioning task. In summary, cortical kindling produces dynamic receptor subunit changes in regions in addition to the seizure focus.

  8. Sucrose ingestion induces rapid AMPA receptor trafficking. (United States)

    Tukey, David S; Ferreira, Jainne M; Antoine, Shannon O; D'amour, James A; Ninan, Ipe; Cabeza de Vaca, Soledad; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K; Hartner, Diana T; Guarini, Carlo B; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F; Khatri, Megna; Marzan, Dave S; Mahajan, Shahana S; Wang, Jing; Froemke, Robert C; Carr, Kenneth D; Aoki, Chiye; Ziff, Edward B


    The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

  9. Exendin-4 promotes the membrane trafficking of the AMPA receptor GluR1 subunit and ADAM10 in the mouse neocortex. (United States)

    Ohtake, Nobuaki; Saito, Mieko; Eto, Masaaki; Seki, Kenjiro


    Glucagon-like peptide-1 (GLP-1) is a novel treatment modality for type 2 diabetes mellitus. However, GLP-1 has been suggested as a therapeutic target for Alzheimer's disease (AD). In rodent studies, GLP-1 reduces amyloid beta (Aβ) and facilitates synaptic plasticity. Therefore, in the present study, we investigated how GLP-1 facilitates synaptic plasticity and reduces the Aβ in vivo. Exendin-4, a GLP-1 receptor agonist that can cross the blood brain barrier, was subcutaneously administered to adult mice. We then extracted the total and the plasma membrane proteins from the mouse neocortex. Exendin-4 significantly increased the phosphorylation level of cAMP response element-binding protein (CREB). Consistently, the expression level of brain-derived neurotrophic factor (BDNF), a transcriptional target of CREB, was increased. Furthermore, exendin-4 increased the membrane protein level of the AMPA receptor GluR1 subunit and postsynaptic density protein-95 (PSD-95), whereas GluR2 was unaffected. These exendin-4-dependent increases in membrane GluR1, total PSD-95 and BDNF were abrogated by pretreatment with temozolomide (TMZ), a DNA-alkylating agent, indicating that these alterations were dependent on exendin-4-induced transcriptional activity. In addition, we found that exendin-4 increased the level of the α-C terminal fragment (α-CTF) of amyloid precursor protein (APP). Furthermore, protein levels of both mature and immature ADAM10, the α-secretase of APP in the plasma membrane, were increased, whereas the total mature and immature ADAM10 levels were unchanged. These exendin-4-dependent increases in α-CTF and ADAM10 were not affected by TMZ. These findings suggested that GLP-1 facilitates the GluR1 membrane insertion through CREB activation and increases α-secretase activity through ADAM10 membrane trafficking. Upregulation of GluR1 and ADAM10 at the plasma membrane were also observed in mice with intracerebroventricular administration of Aβ oligomer

  10. ABP: a novel AMPA receptor binding protein. (United States)

    Srivastava, S; Ziff, E B


    We review the cloning of a novel AMPA receptor binding protein (ABP) that interacts with GluR2/3 and is homologous to GRIP. ABP is enriched in the PSD with GluR2 and is localized to the PSD by EM. ABP binds GluR2 via the C-terminal VXI motif through a Class I PDZ interaction. ABP and GRIP can also homo- and heteromultimerize. Thus, ABP and GRIP may be involved in AMPA receptor regulation and localization, by linking it to other cytoskeletal or signaling molecules. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors. We are currently investigating proteins that bind ABP and that may regulate the AMPA receptor.

  11. Agonist discrimination between AMPA receptor subtypes

    DEFF Research Database (Denmark)

    Coquelle, T; Christensen, J K; Banke, T G


    The lack of subtype-selective compounds for AMPA receptors (AMPA-R) led us to search for compounds with such selectivity. Homoibotenic acid analogues were investigated at recombinant GluR1o, GluR2o(R), GluR3o and GluR1o + 3o receptors expressed in Sf9 insect cells and affinities determined in [3H...

  12. Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant. (United States)

    Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce


    Previously, we proposed a two-stage model for an in vitro neural correlate of eyeblink classical conditioning involving the initial synaptic incorporation of glutamate receptor A1 (GluA1)-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type receptors (AMPARs) followed by delivery of GluA4-containing AMPARs that support acquisition of conditioned responses. To test specific elements of our model for conditioning, selective knockdown of GluA4 AMPAR subunits was used using small-interfering RNAs (siRNAs). Recently, we sequenced and characterized the GluA4 subunit and its splice variants from pond turtles, Trachemys scripta elegans (tGluA4). Analysis of the relative abundance of mRNA expression by real-time RT-PCR showed that the flip/flop variants of tGluA4, tGluA4c, and a novel truncated variant tGluA4trc1 are major isoforms in the turtle brain. Here, transfection of in vitro brain stem preparations with anti-tGluA4 siRNA suppressed conditioning, tGluA4 mRNA and protein expression, and synaptic delivery of tGluA4-containing AMPARs but not tGluA1 subunits. Significantly, transfection of abducens motor neurons by nerve injections of tGluA4 flop rescue plasmid prior to anti-tGluA4 siRNA application restored conditioning and synaptic incorporation of tGluA4-containing AMPARs. In contrast, treatment with rescue plasmids for tGluA4 flip or tGluA4trc1 failed to rescue conditioning. Finally, treatment with a siRNA directed against GluA1 subunits inhibited conditioning and synaptic delivery of tGluA1-containing AMPARs and importantly, those containing tGluA4. These data strongly support our two-stage model of conditioning and our hypothesis that synaptic incorporation of tGluA4-containing AMPARs underlies the acquisition of in vitro classical conditioning. Furthermore, they suggest that tGluA4 flop may have a critical role in conditioning mechanisms compared with the other tGluA4 splice variants.

  13. Effects of cyclothiazide on GluR1/AMPA receptors (United States)

    Fucile, Sergio; Miledi, Ricardo; Eusebi, Fabrizio


    Cyclothiazide (CTZ), a positive allosteric modulator of ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, is used frequently to block the desensitization of both native and heterologously expressed AMPA receptors. Specifically, CTZ is known to produce a fast inhibition of AMPA receptor desensitization and a much slower potentiation of the AMPA current. By using patch-clamp techniques, the effects of CTZ were studied in HEK 293 cells stably transfected with the rat flip GluR1 subunit. Upon CTZ treatment, we found an increased apparent affinity for the agonist, a slow whole-cell current potentiation, a fast inhibition of desensitization, and a lengthening of single-channel openings. Furthermore, we show that CTZ alters the channel gating events modifying the relative contribution of different single-channel classes of conductance (γ), increasing and decreasing, respectively, the contributions of γM (medium) and γL (low) without altering that of the γH (high) conductance channels. We also present a kinetic model that predicts well all of the experimental findings of CTZ action. Finally, we suggest a protocol for standard cell treatment with CTZ to attain maximal efficacy of CTZ on GluR1 receptors. PMID:16473938

  14. AMPA receptor inhibition by synaptically released zinc. (United States)

    Kalappa, Bopanna I; Anderson, Charles T; Goldberg, Jacob M; Lippard, Stephen J; Tzounopoulos, Thanos


    The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.

  15. Inhibitory interneuron classes express complementary AMPA-receptor patterns in macaque primary visual cortex

    NARCIS (Netherlands)

    Kooijmans, Roxana N; Self, Matthew W; Wouterlood, Floris G; Beliën, Jeroen A M; Roelfsema, Pieter R


    Glutamate receptors mediate excitatory neurotransmission. A very prevalent type of glutamate receptor in the neocortex is the AMPA receptor (AMPAR). AMPARs mediate fast synaptic transmission and their functionality depends on the subunit composition. In primary visual cortex (area V1), the density a

  16. Ganglioside Regulation of AMPA Receptor Trafficking (United States)

    Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.


    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868

  17. Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus. (United States)

    Castilho, A F; Liberal, J T; Baptista, F I; Gaspar, J M; Carvalho, A L; Ambrósio, A F


    Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

  18. DHEAS increases levels of GluR2/3 and GluR2, AMPA receptor subunits, in C57BL/6 mice hippocampus El DHEAS incrementa la expresión de GluR2/3 y GLUR2 del receptor AMPA en el hipocampo de ratones C57/BL6

    Directory of Open Access Journals (Sweden)

    Diego Sepúlveda Falla


    Full Text Available

    Dehydroepiandrosterone sulfate (DHEA-S is a neurosteroid that has effects such as neuromodulator of synaptic transmission and neuroprotection. The specific signaling pathways for these effects are not elucidated yet. Given that, some neurosteroids act through the activation of ionotropic glutamate receptors, therefore the effect of DHEA-S on the subunits GluR2  and GluR3 of the AMPA receptor was evaluated.  Either DHEA-S or a control substance was administered to C57/BL6 mice. Subunit expression of the AMPA receptor was analyzed by Western blotting.



    Results show that long-term DHEA-S administration to C57/BL6 mice, increases the protein levels of the subunits GluR2 and GluR2/3 of the AMPA receptors located in the hippocampus.

  19. Ganglioside regulation of AMPA receptor trafficking. (United States)

    Prendergast, Jillian; Umanah, George K E; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G; Cole, Robert N; Huganir, Richard L; Dawson, Ted M; Dawson, Valina L; Schnaar, Ronald L


    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.

  20. Alterations in Hippocampal Oxidative Stress, Expression of AMPA Receptor GluR2 Subunit and Associated Spatial Memory Loss by Bacopa monnieri Extract (CDRI-08) in Streptozotocin-Induced Diabetes Mellitus Type 2 Mice. (United States)

    Pandey, Surya P; Singh, Hemant K; Prasad, S


    Bacopa monnieri extract has been implicated in the recovery of memory impairments due to various neurological disorders in animal models and humans. However, the precise molecular mechanism of the role of CDRI-08, a well characterized fraction of Bacopa monnieri extract, in recovery of the diabetes mellitus-induced memory impairments is not known. Here, we demonstrate that DM2 mice treated orally with lower dose of CDRI-08 (50- or 100 mg/kg BW) is able to significantly enhance spatial memory in STZ-DM2 mice and this is correlated with a significant decline in oxidative stress and up regulation of the AMPA receptor GluR2 subunit gene expression in the hippocampus. Treatment of DM2 mice with its higher dose (150 mg/kg BW or above) shows anti-diabetic effect in addition to its ability to recover the spatial memory impairment by reversing the DM2-induced elevated oxidative stress and decreased GluR2 subunit expression near to their values in normal and CDRI-08 treated control mice. Our results provide evidences towards molecular basis of the memory enhancing and anti diabetic role of the Bacopa monnieri extract in STZ-induced DM2 mice, which may have therapeutic implications.

  1. Perampanel inhibition of AMPA receptor currents in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Chao-Yin Chen

    Full Text Available Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM, concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×105 M-1 s-1 and 0.58 s-1, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC50, 0.56 µM was similar at all kainate concentrations (3-100 µM, demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics.

  2. Nuclear respiratory factor 1 co-regulates AMPA glutamate receptor subunit 2 and cytochrome c oxidase: tight coupling of glutamatergic transmission and energy metabolism in neurons. (United States)

    Dhar, Shilpa S; Liang, Huan Ling; Wong-Riley, Margaret T T


    Neuronal activity, especially of the excitatory glutamatergic type, is highly dependent on energy from the oxidative pathway. We hypothesized that the coupling existed at the transcriptional level by having the same transcription factor to regulate a marker of energy metabolism, cytochrome c oxidase (COX) and an important subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors, GluR2 (Gria2). Nuclear respiratory factor 1 (NRF-1) was a viable candidate because it regulates all COX subunits and potentially activates Gria2. By means of in silico analysis, electrophoretic mobility shift and supershift, chromatin immunoprecipitation, and promoter mutational assays, we found that NRF-1 functionally bound to Gria2 promoter. Silencing of NRF-1 with small interference RNA prevented the depolarization-stimulated up-regulation of Gria2 and COX, and over-expression of NRF-1 rescued neurons from tetrodotoxin-induced down-regulation of Gria2 and COX transcripts. Thus, neuronal activity and energy metabolism are tightly coupled at the molecular level, and NRF-1 is a critical agent in this process.

  3. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits.

    Directory of Open Access Journals (Sweden)

    Suleman Hussain

    Full Text Available Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density. We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2 and glutamate receptor subunit 1 (GluA1. Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.

  4. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits. (United States)

    Hussain, Suleman; Davanger, Svend


    Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.

  5. Ontogeny of AMPA and NMDA receptor gene expression in the developing sheep white matter and cerebral cortex. (United States)

    Dean, Justin M; Fraser, Mhoyra; Shelling, Andrew N; Bennet, Laura; George, Sherly; Shaikh, Shamim; Scheepens, Arjan; Gunn, Alistair J


    This study examined the hypothesis that the high prevalence of white matter injury in premature infants is associated with increased expression of calcium-permeable forms of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors in pre-myelinating white matter. We characterized expression of subunits of the AMPA, and for reference, the N-methyl-d-aspartate (NMDA), glutamate receptors at 0.5, 0.65, 0.85, and term gestation in the ovine fetal white matter and cerebral cortex. There was a low expression of the critical calcium-impermeable AMPA receptor GluR2 subunit in subcortical white matter both absolutely and relative to other AMPA subunits throughout gestation. In contrast, GluR2 subunit mRNA expression fell in the cerebral cortex with increasing gestation whereas protein expression increased. These findings suggest a vulnerability of subcortical white matter to AMPA receptor-mediated calcium toxicity throughout the second half of gestation. Thus, the hypothesis that AMPA receptor-mediated glutamate toxicity contributes to brain damage in premature infants needs to be revised.

  6. Alternative Splicing of AMPA subunits in Prefrontal Cortical Fields of Cynomolgus Monkeys following Chronic Ethanol Self-Administration

    Directory of Open Access Journals (Sweden)

    Glen eAcosta


    Full Text Available Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in alcohol dependence. To this end, the effects of chronic ethanol self-administration on glutamate receptor ionotropic AMPA (GRIA subunit variant and kainate (GRIK subunit mRNA expression were studied in the orbitofrontal cortex (OFC, dorsolateral prefrontal cortex (DLPFC and anterior cingulate cortex (ACC of male cynomolgus monkeys. In DLPFC, total AMPA splice variant expression and total kainate receptor subunit expression were significantly decreased in alcohol drinking monkeys. Expression levels of GRIA3 flip and flop and GRIA4 flop mRNAs in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. In OFC, AMPA subunit splice variant expression was reduced in the alcohol treated group. GRIA2 flop mRNA levels in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. Results from these studies provide further evidence of transcriptional regulation of iGluR subunits in the primate brain following chronic alcohol self-administration. Additional studies examining the cellular localization of such effects in the framework of primate prefrontal cortical circuitry are warranted.

  7. Novel bivalent positive allosteric modulators of AMPA receptor. (United States)

    Lavrov, M I; Grigor'ev, V V; Bachurin, S O; Palyulin, V A; Zefirov, N S


    A positive allosteric modulator of AMPA receptors has been designed using computer-aided molecular modeling techniques. It possessed a record high experimentally confirmed potency in the picomolar concentration range and belongs to a new type of bivalent AMPA receptor ligands containing bicyclo[3.3.1]nonane scaffold. The suggested structure could serve as a basis for further optimization and development of drugs for the treatment of neurodegenerative diseases, cognition enhancement, and improvement of memory.

  8. 内源性大麻素对海马神经元 AMPA 受体GluR2的作用%Effect of endocannabinoids pretreatment on the expression of AMPA receptor GluR2 subunit in the hippocampal pyramidal neurons of mouse

    Institute of Scientific and Technical Information of China (English)

    刘曌宇; 高杨; 孙思斯; 陈绍洋; 王强


    目的:探索内源性大麻素预处理对小鼠全脑缺血再灌注损伤后海马神经元α-氨基-3羟基-5-甲基-4-异恶唑丙酸(AMPA)受体2亚基(GluR2)表达的影响及机制。方法雄性C57BL/6小鼠随机分为假手术组、模型组、2-花生酰基甘油(2-AG)处理组、大麻素1型受体( CB1R)拮抗剂( AM251)+2-AG组和溶剂组。2-AG 处理组腹腔注射2-AG 5 mg/kg;AM251+2-AG 组腹腔注射AM2511 mg/kg,30 min后腹腔给予2-AG 5 mg/kg;溶剂组腹腔给予0.1 ml二甲基亚砜( DMSO)。各组小鼠在预处理后30 min采用夹闭双侧颈总动脉20 min行再灌注的方法制备全脑缺血再灌注损伤模型。再灌注2 h取材行Western blot及免疫荧光检测。结果 GluR2高表达于正常小鼠海马CA1区锥体神经元;C57小鼠全脑缺血20 min,再灌后2 h海马组织GluR2表达明显下调(P<0.05);与模型组比较,2-AG处理组海马组织GluR2明显增高(P<0.05);与2-AG处理组比较,AM251+2-AG组海马组织GluR2明显下降(P<0.05)。结论内源性大麻素2-AG作用于神经元CB1R,逆转全脑缺血损伤导致的海马神经元GluR2表达下降。%Objective Whether endocannabinoids pretreatment affects the expression of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor 2 subunit (GluR2) in mice after global cerebral ischemia-reperfusion (I/R) injury and its mechanism are investigated.Methods Adult male C57BL/6 mice were randomly divided into five groups:sham group, I/R group, 2-arachidonoyl glycerol (2-AG) group, cannabinoid 1 receptor (CB1R) antagonist(AM251)+2-AG group and vehicle group.The mice in 2-AG group were injected 2-AG with a dose of 5 mg/kg intraperitoneally.AM251 was injected intraperitoneally with a dose of 1 mg/kg 30 min before pretreatment.2-AG was administered in AM251+2-AG group mice with a dose of 5 mg/kg.A total of 0.1 ml Dimethylsulfoxide (DMSO

  9. 3-Substituted phenylalanines as selective AMPA- and kainate receptor ligands

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Pickering, Darryl S; Nielsen, Birgitte;


    On the basis of X-ray structures of ionotropic glutamate receptor constructs in complex with amino acid-based AMPA and kainate receptor antagonists, a series of rigid as well as flexible biaromatic alanine derivatives carrying selected hydrogen bond acceptors and donors have been synthesized in o...

  10. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.


    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  11. Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters. (United States)

    DeSouza, Sunita; Fu, Jie; States, Bradley A; Ziff, Edward B


    Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

  12. Actin-dependent mechanisms in AMPA receptor trafficking

    Directory of Open Access Journals (Sweden)

    Jonathan G Hanley


    Full Text Available The precise regulation of AMPA receptor (AMPAR number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits during learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signalling pathways that modulate actin polymerization and depolymerisation. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine.

  13. Are AMPA Receptor Positive Allosteric Modulators Potential Pharmacotherapeutics for Addiction?

    Directory of Open Access Journals (Sweden)

    Lucas R. Watterson


    Full Text Available Positive allosteric modulators (PAMs of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications.

  14. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes (United States)

    Ruiz, A; Matute, C; Alberdi, E


    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  15. AMPA receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization

    DEFF Research Database (Denmark)

    Jensen, J B; Schousboe, A; Pickering, D S;


    was blocked was seen as early as 5 DIV since 10 microM MK-801 did not completely block the response whereas 10 microM NBQX did. The 2,3-benzodiazepine GYKI compounds, which have been reported to be selective non-competitive AMPA receptor antagonists, were here observed to block the AMPA toxicity...

  16. Ceftriaxone attenuates cocaine relapse after abstinence through modulation of nucleus accumbens AMPA subunit expression. (United States)

    LaCrosse, Amber L; Hill, Kristine; Knackstedt, Lori A


    Using the extinction-reinstatement model of cocaine relapse, we and others have demonstrated that the antibiotic ceftriaxone attenuates cue- and cocaine-primed reinstatement of cocaine-seeking. Reinstatement is contingent on the release of glutamate in the nucleus accumbens core (NAc) and manipulations that reduce glutamate efflux or block post-synaptic glutamate receptors attenuate reinstatement. We have demonstrated that the mechanism of action by which ceftriaxone attenuates reinstatement involves increased NAc GLT-1 expression and a reduction in NAc glutamate efflux during reinstatement. Here we investigated the effects of ceftriaxone (100 and 200 mg/kg) on context-primed relapse following abstinence without extinction training and examined the effects of ceftriaxone on GluA1, GluA2 and GLT-1 expression. We conducted microdialysis during relapse to determine if an increase in NAc glutamate accompanies relapse after abstinence and whether ceftriaxone blunts glutamate efflux. We found that both doses of ceftriaxone attenuated relapse. While relapse was accompanied by an increase in NAc glutamate, ceftriaxone (200 mg/kg) was unable to significantly reduce NAc glutamate efflux during relapse despite its ability to upregulate GLT-1. GluA1 was reduced in the NAc by both doses of ceftriaxone while GluA2 expression was unchanged, indicating that ceftriaxone altered AMPA subunit composition following cocaine. Finally, GLT-1 was not altered in the PFC by ceftriaxone. These results indicate that it is possible to attenuate context-primed relapse to cocaine-seeking through modification of post-synaptic receptor properties without attenuating glutamate efflux during relapse. Furthermore, increasing NAc GLT-1 protein expression is not sufficient to attenuate glutamate efflux.

  17. Activity-Dependent Ubiquitination of GluA1 and GluA2 Regulates AMPA Receptor Intracellular Sorting and Degradation


    Jocelyn Widagdo; Ye Jin Chai; Margreet C. Ridder; Yu Qian Chau; Richard C. Johnson; Pankaj Sah; Richard L. Huganir; Victor Anggono


    AMPA receptors (AMPARs) have recently been shown to undergo post-translational ubiquitination in mammalian neurons. However, the underlying molecular mechanisms are poorly understood and remain controversial. Here, we report that all four AMPAR subunits (GluA1-4) are rapidly ubiquitinated upon brief application of AMPA or bicuculline in cultured neurons. This process is Ca2+ dependent and requires the activity of L-type voltage-gated Ca2+ channels and Ca2+/calmodulin-dependent kinase II. The ...

  18. AMPA receptor potentiation can prevent ethanol-induced intoxication. (United States)

    Jones, Nicholas; Messenger, Marcus J; O'Neill, Michael J; Oldershaw, Anna; Gilmour, Gary; Simmons, Rosa M A; Iyengar, Smriti; Libri, Vincenzo; Tricklebank, Mark; Williams, Steve C R


    We present a substantial series of behavioral and imaging experiments, which demonstrate, for the first time, that increasing AMPA receptor-mediated neurotransmission via administration of potent and selective biarylsulfonamide AMPA potentiators LY404187 and LY451395 reverses the central effects of an acutely intoxicating dose of ethanol in the rat. Using pharmacological magnetic resonance imaging (phMRI), we observed that LY404187 attenuated ethanol-induced reductions in blood oxygenation level dependent (BOLD) in the anesthetized rat brain. A similar attenuation was apparent when measuring local cerebral glucose utilization (LCGU) via C14-2-deoxyglucose autoradiography in freely moving conscious rats. Both LY404187 and LY451395 significantly and dose-dependently reversed ethanol-induced deficits in both motor coordination and disruptions in an operant task where animals were trained to press a lever for food reward. Both prophylactic and acute intervention treatment with LY404187 reversed ethanol-induced deficits in motor coordination. Given that LY451395 and related AMPA receptor potentiators/ampakines are tolerated in both healthy volunteers and elderly patients, these data suggest that such compounds may form a potential management strategy for acute alcohol intoxication.

  19. Parvalbumin-containing interneurons in rat hippocampus have an AMPA receptor profile suggestive of vulnerability to excitotoxicity. (United States)

    Moga, Diana; Hof, Patrick R; Vissavajjhala, Prabhakar; Moran, Thomas M; Morrison, John H


    alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate excitatory neurotransmission in the central nervous system, and contain combinations of four subunits (GluR1-4). We developed a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and most interneurons. In addition, we found a subset of parvalbumin (PV)-containing interneurons in the hippocampus and neocortex that was notable for its intense GluR3 immunoreactivity and lack of GluR2 immunoreactivity. Such an expression pattern of AMPA receptor subunits is likely to make these interneurons selectively vulnerable to excitotoxicity.

  20. Phenotype of mice with inducible ablation of GluA1 AMPA receptors during late adolescence: relevance for mental disorders. (United States)

    Inta, Dragos; Vogt, Miriam A; Elkin, Hasan; Weber, Tillmann; Lima-Ojeda, Juan M; Schneider, Miriam; Luoni, Alessia; Riva, Marco A; Gertz, Karen; Hellmann-Regen, Julian; Kronenberg, Golo; Meyer-Lindenberg, Andreas; Sprengel, Rolf; Gass, Peter


    Adolescence is characterized by important molecular and anatomical changes with relevance for the maturation of brain circuitry and cognitive function. This time period is of critical importance in the emergence of several neuropsychiatric disorders accompanied by cognitive impairment, such as affective disorders and schizophrenia. The molecular mechanisms underlying these changes at neuronal level during this specific developmental stage remains however poorly understood. GluA1-containing AMPA receptors, which are located predominantly on hippocampal neurons, are the primary molecular determinants of synaptic plasticity. We investigated here the consequences of the inducible deletion of GluA1 AMPA receptors in glutamatergic neurons during late adolescence. We generated mutant mice with a tamoxifen-inducible deletion of GluA1 under the control of the CamKII promoter for temporally and spatially restricted gene manipulation. GluA1 ablation during late adolescence induced cognitive impairments, but also marked hyperlocomotion and sensorimotor gating deficits. Unlike the global genetic deletion of GluA1, inducible GluA1 ablation during late adolescence resulted in normal sociability. Deletion of GluA1 induced redistribution of GluA2 subunits, suggesting AMPA receptor trafficking deficits. Mutant animals showed increased hippocampal NMDA receptor expression and no change in striatal dopamine concentration. Our data provide new insight into the role of deficient AMPA receptors specifically during late adolescence in inducing several cognitive and behavioral alterations with possible relevance for neuropsychiatric disorders.

  1. DCP-LA stimulates AMPA receptor exocytosis through CaMKII activation due to PP-1 inhibition. (United States)

    Kanno, Takeshi; Yaguchi, Takahiro; Nagata, Tetsu; Tanaka, Akito; Nishizaki, Tomoyuki


    The linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) by inhibiting protein phosphatase-1 (PP-1). DCP-LA induced a transient huge facilitation of synaptic transmission monitored from the CA1 region of rat hippocampal slices, which was largely inhibited by the CaMKII inhibitor KN-93. DCP-LA potentiated kainate-evoked whole-cell membrane currents for Xenopus oocytes expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors composed of the GluR1, GluR3, GluR1/GluR2, GluR1/GluR3, and GluR1/GluR2/GluR3 subunits, and the potentiation was significantly inhibited by KN-93. A similar potentiation was still found with mutant GluR1 (S831A) receptor lacking CaMKII phosphorylation site. The GluR1 and GluR2 subunits formed AMPA receptors in the rat hippocampus, and DCP-LA increased expression of both the subunits on the plasma membrane. The DCP-LA action was blocked by KN-93 and the exocytosis inhibitor botulinum toxin type A, but not by the endocytosis inhibitor phenylarsine oxide. DCP-LA, thus, appears to activate CaMKII through PP-1 inhibition, that stimulates AMPA receptor exocytosis to increase expression of the receptors on the plasma membrane, responsible for potentiate AMPA receptor responses and facilitation of hippocampal synaptic transmission.

  2. Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamatergic synapses. (United States)

    Wang, Xulong; Lippi, Giordano; Carlson, David M; Berg, Darwin K


    Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert 'silent' glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. We find that activation of nicotinic receptors on astrocytes releases a component that specifically recruits AMPA receptors to glutamatergic synapses. The recruitment appears to occur preferentially at what may be 'silent synapses', that is, synapses that have all the components required for glutamatergic transmission (including NMDA receptors) but lack sufficient AMPA receptors to generate a response. The results are unexpected and open up new possibilities for mechanisms underlying network formation and synaptic plasticity.

  3. The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors. (United States)

    Wang, Rui; Mellem, Jerry E; Jensen, Michael; Brockie, Penelope J; Walker, Craig S; Hoerndli, Frédéric J; Hauth, Linda; Madsen, David M; Maricq, Andres V


    The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

  4. Estudio computacional de las relaciones evolutivas de los receptores ionotrópicos NMDA, AMPA y kainato en cuatro especies de primates

    Directory of Open Access Journals (Sweden)

    Francy Johanna Moreno-Pedraza


    Full Text Available Computational study of the evolutionary relationships of the ionotropic receptors NMDA, AMPA and kainate in four species ofprimates. Objective. To identify the influence of changes on the secondary structure and evolutionary relationship of NMDA, AMPA andkainate receptors in Homo sapiens, Pan troglodytes, Pongo pygmaeus and Macaca mulatta. Materials and methods. We identified 91sequences for NMDA, AMPA and kainate receptors and analyzed with software for predicting secondary structure, phosphorylation sites,multiple alignments, selection of protein evolution models and phylogenetic prediction. Results. We found that subunits GLUR5, NR2A,NR2C and NR3A showed structural changes in the C-terminal region and formation or loss of phosphorylation sites in this zone.Additionally the phylogenetic prediction suggests that the NMDA NR2 subunits are the closest to the ancestral node that gives rise to theother subunits. Conclusions. Changes in structure and phosphorylation sites in GLUR5, NR2A, NR2C and NR3A subunits suggestvariations in the interaction of the C-terminal region with kinase proteins and with proteins with PDZ domains, which could affect thetrafficking and anchoring of the subunits. On the other hand, the phylogenetic prediction suggests that the changes that occurred in the NR2subunits gave rise to the other subunits of glutamate ionotropic receptors, primarily because the NMDA and particularly the NR2D subunitsare the most closely related to the ancestral node that possibly gave rise to the iGluRs.

  5. Impaired associative fear learning in mice with complete loss or haploinsufficiency of AMPA GluR1 receptors

    Directory of Open Access Journals (Sweden)

    Michael Feyder


    Full Text Available There is compelling evidence that L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA glutamate receptors containing the GluR1 subunit contribute to the molecular mechanisms associated with learning. AMPA GluR1 glutamate receptor knockout mice (KO exhibit abnormal hippocampal and amygdala plasticity, and deficits on various assays for cognition including Pavlovian fear conditioning. Here we examined associative fear learning in mice with complete absence (KO or partial loss (heterozygous mutant, HET of GluR1 on multiple fear conditioning paradigms. After multi-trial delay or trace conditioning, KO displayed impaired tone and context fear recall relative to WT, whereas HET were normal. After one-trial delay conditioning, both KO and HET showed impaired tone and context recall. HET and KO showed normal nociceptive sensitivity in the hot plate and tail flick tests. These data demonstrate that the complete absence of GluR1 subunit-containing receptors prevents the formation of associative fear memories, while GluR1 haploinsufficiency is sufficient to impair one-trial fear learning. These findings support growing evidence of a major role for GluR1-containing AMPA receptors in amygdalamediated forms of learning and memory.

  6. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

    DEFF Research Database (Denmark)

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L;


    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling...

  7. Stereostructure-activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Johansen, Tommy N; Greenwood, Jeremy R; Frydenvang, Karla Andrea;


    -methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of these binding domain crystal structures has formed the basis for rational...... design of ligands, especially for the AMPA and kainate subtypes of ionotropic Glu receptors. This mini-review will focus on structure-activity relationships on AMPA and kainate receptor agonists with special emphasis on stereochemical and three-dimensional aspects....

  8. mTOR is essential for corticosteroid effects on hippocampal AMPA receptor function and fear memory

    NARCIS (Netherlands)

    Xiong, H.; Cassé, F.; Zhou, Y.; Zhou, M.; Xiong, Z.Q.; Joëls, M.; Martin, S.; Krugers, H.J.


    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptors (AMPARs), which are crucial for synaptic plasticity and memory formation. Combining a live imaging flu

  9. Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors.


    Dennis, Siobhan; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G.; Henley, Jeremy M.; Mellor, Jack R.


    Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensi...

  10. Oxygen/glucose Deprivation Induces a Reduction in Synaptic AMPA Receptors on Hippocampal CA3 Neurons Mediated by mGluR1 and A3 Receptors


    Dennis, Siobhan H.; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G.; Henley, Jeremy M.; Mellor, Jack R.


    Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighbouring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+ resulting in delayed cell death. However, it is unclear if the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivit...

  11. Ionotropic excitatory amino acid receptor ligands. Synthesis and pharmacology of a new amino acid AMPA antagonist

    DEFF Research Database (Denmark)

    Madsen, U; Sløk, F A; Stensbøl, T B;


    We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4......-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic...... excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2...

  12. Rational Design of a Novel AMPA Receptor Modulator through a Hybridization Approach (United States)


    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a family of glutamate ion channels of considerable interest in excitatory neurotransmission and associated disease processes. Here, we demonstrate how exploitation of the available X-ray crystal structure of the receptor ligand binding domain enabled the development of a new class of AMPA receptor positive allosteric modulators (7) through hybridization of known ligands (5 and 6), leading to a novel chemotype with promising pharmacological properties. PMID:25893038

  13. Hormonal regulation of AMPA receptor trafficking and memory formation

    Directory of Open Access Journals (Sweden)

    Harmen J Krugers


    Full Text Available Humans and rodents retain memories for stressful events very well. The facilitated retention of these memories is normally very useful. However, in susceptible individuals a variety of pathological conditions may develop in which memories related to stressful events remain inappropriately present, such as in post-traumatic stress disorder. The memory enhancing effects of stress are mediated by hormones, such as norepinephrine and glucocorticoids which are released during stressful experiences. Here we review recently identified molecular mechanisms that underlie the effects of stress hormones on synaptic efficacy and learning and memory. We discuss AMPA receptors as major target for stress hormones and describe a model in which norepinephrine and glucocorticoids are able to strengthen and prolong different phases of stressful memories.

  14. An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors.

    Directory of Open Access Journals (Sweden)

    Morgane Riou

    Full Text Available NMDA receptors (NMDARs form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular and functional (plasma-membrane inserted pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.

  15. Synaptic transmission and plasticity require AMPA receptor anchoring via its N-terminal domain (United States)

    Watson, Jake F; Ho, Hinze; Greger, Ingo H


    AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and are selectively recruited during activity-dependent plasticity to increase synaptic strength. A prerequisite for faithful signal transmission is the positioning and clustering of AMPARs at postsynaptic sites. The mechanisms underlying this positioning have largely been ascribed to the receptor cytoplasmic C-termini and to AMPAR-associated auxiliary subunits, both interacting with the postsynaptic scaffold. Here, using mouse organotypic hippocampal slices, we show that the extracellular AMPAR N-terminal domain (NTD), which projects midway into the synaptic cleft, plays a fundamental role in this process. This highly sequence-diverse domain mediates synaptic anchoring in a subunit-selective manner. Receptors lacking the NTD exhibit increased mobility in synapses, depress synaptic transmission and are unable to sustain long-term potentiation (LTP). Thus, synaptic transmission and the expression of LTP are dependent upon an AMPAR anchoring mechanism that is driven by the NTD. DOI: PMID:28290985

  16. Calcium-permeable AMPA receptors in the nucleus accumbens regulate depression-like behaviors in the chronic neuropathic pain state. (United States)

    Goffer, Yossef; Xu, Duo; Eberle, Sarah E; D'amour, James; Lee, Michelle; Tukey, David; Froemke, Robert C; Ziff, Edward B; Wang, Jing


    Depression is a salient emotional feature of chronic pain. Depression alters the pain threshold and impairs functional recovery. To date, however, there has been limited understanding of synaptic or circuit mechanisms that regulate depression in the pain state. Here, we demonstrate that depression-like behaviors are induced in a rat model of chronic neuropathic pain. Using this model, we show that chronic pain selectively increases the level of GluA1 subunits of AMPA-type glutamate receptors at the synapses of the nucleus accumbens (NAc), a key component of the brain reward system. We find, in addition, that this increase in GluA1 levels leads to the formation of calcium-permeable AMPA receptors (CPARs). Surprisingly, pharmacologic blockade of these CPARs in the NAc increases depression-like behaviors associated with pain. Consistent with these findings, an AMPA receptor potentiator delivered into the NAc decreases pain-induced depression. These results show that transmission through CPARs in the NAc represents a novel molecular mechanism modulating the depressive symptoms of pain, and thus CPARs may be a promising therapeutic target for the treatment of pain-induced depression. More generally, these findings highlight the role of central glutamate signaling in pain states and define the brain reward system as an important region for the regulation of depressive symptoms of pain.

  17. Effects of Food Restriction and Sucrose Intake on Synaptic Delivery of AMPA Receptors in Nucleus Accumbens


    Peng, Xing-Xiang; Ziff, Edward B.; Carr, Kenneth D.


    Insertion and removal of AMPA receptors from the synaptic membrane underlie dynamic tuning of synaptic transmission and enduring changes in synaptic strength. Preclinical addiction research suggests that AMPA receptor trafficking plays an important role in nucleus accumbens (NAc) neuroplasticity underlying the compulsive and persistent quality of drug-seeking. Considering the parallels between drug addiction and compulsive eating, plus the supranormal reward properties of sucrose, and the rol...

  18. Differential roles for NSF and GRIP/ABP in AMPA receptor cycling. (United States)

    Braithwaite, Steven P; Xia, Houhui; Malenka, Robert C


    alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) stability and movement at synapses are important factors controlling synaptic strength. Here, we study the roles of proteins [N-ethylmaleimide-sensitive fusion protein (NSF), glutamate receptor AMPAR binding protein (ABP)-interacting protein (GRIP)/(ABP), and protein interacting with C-kinase-1 (PICK1) that interact with the GluR2 subunit in the control of the surface expression and cycling of AMPARs. Epitope-tagged GluR2 formed functional receptors that exhibited targeting to synaptic sites. Constructs in which binding to NSF, PDZ proteins (GRIP/ABP and PICK1), or GRIP/ABP alone was eliminated each exhibited normal surface targeting and constitutive cycling. The lack of NSF binding, however, resulted in receptors that were endocytosed to a greater extent than wild-type receptors in response to application of AMPA or N-methyl-d-aspartate (NMDA). Conversely, the behavior of the GluR2 mutants incapable of binding to GRIP/ABP suggests that these PDZ proteins play a role in the stabilization of an intracellular pool of AMPARs that have been internalized on stimulation, thus inhibiting their recycling to the synaptic membrane. These results provide further evidence for distinct functional roles of GluR2-interacting proteins in AMPAR trafficking.

  19. Differential effects of two chronic diazepam treatment regimes on withdrawal anxiety and AMPA receptor characteristics. (United States)

    Allison, Claire; Pratt, Judith A


    Withdrawal from chronic benzodiazepines is associated with increased anxiety and seizure susceptibility. Neuroadaptive changes in neural activity occur in limbo-cortical structures although changes at the level of the GABA(A) receptor do not provide an adequate explanation for these functional changes. We have employed two diazepam treatment regimes known to produce differing effects on withdrawal aversion in the rat and examined whether withdrawal-induced anxiety was accompanied by changes in AMPA receptor characteristics. Rats were given 28 days treatment with diazepam by the intraperitoneal (i.p.) route (5 mg/kg) and the subcutaneous (s.c.) route (15 mg/kg). Withdrawal anxiety in the elevated plus maze was evident in the group withdrawn from chronic s.c. diazepam (relatively more stable plasma levels) but not from the chronic i.p. group (fluctuating daily plasma levels). In the brains of these rats, withdrawal anxiety was accompanied by increased [3H]Ro48 8587 binding in the hippocampus and thalamus, and decreased GluR1 and GluR2 subunit mRNA expression in the amygdala (GluR1 and GluR2) and cortex (GluR1). The pattern of changes was different in the chronic i.p. group where in contrast to the chronic s.c. group, there was reduced [3H]Ro48 8587 binding in the hippocampus and no alterations in GluR1 and GluR2 subunit expression in the amygdala. While both groups showed reduced GluR1 mRNA subunit expression in the cortex overall, only the agranular insular cortex exhibited marked reductions following chronic i.p. diazepam. Striatal GluR2 mRNA expression was increased in the i.p. group but not the s.c. group. Taken together, these data are consistent with differential neuroadaptive processes in AMPA receptor plasticity being important in withdrawal from chronic benzodiazepines. Moreover, these processes may differ both at a regional and receptor function level according to the behavioral manifestations of withdrawal.

  20. Structural and pharmacological characterization of phenylalanine-based AMPA receptor antagonists at kainate receptors

    DEFF Research Database (Denmark)

    Venskutonyte, Raminta; Frydenvang, Karla; Valadés, Elena Antón;


    . A new series of phenylalanine derivatives that target iGluRs was reported to bind AMPA receptors. Herein we report our studies of these compounds at the kainate receptors GluK1-3. Several compounds bind with micromolar affinity at GluK1 and GluK3, but do not bind GluK2. The crystal structure of the most...

  1. NMDA and AMPA receptors contribute to the maintenance of substance P-induced thermal hyperalgesia. (United States)

    Nakayama, Tomohiro; Naono, Rumi; Ikeda, Tetsuya; Nishimori, Toshikazu


    It is well known that intrathecal administration of substance P (SP) induces thermal hyperalgesia, but the mechanisms underlying the maintenance of SP-induced thermal hyperalgesia remain to be clarified. Thus, to clarify the receptors involved in the maintenance of SP-induced thermal hyperalgesia, the effect of administering SP or glutamate receptor agonists, NMDA or AMPA, under SP-induced thermal hyperalgesia was investigated. Also, the effect of pretreatment with protein kinase inhibitors on scratching behavior by NMDA or AMPA under SP-induced thermal hyperalgesia was examined. Under SP-induced thermal hyperalgesia, the number of scratchings following SP administration was time-dependently suppressed, whereas the number of scratchings after NMDA or AMPA administration was markedly enhanced and SP-induced thermal hyperalgesia was attenuated by pretreatment with NMDA or AMPA receptor antagonist. Furthermore, pretreatment with kinase inhibitors significantly attenuated the enhancement of scratching behavior by NMDA or AMPA under SP-induced thermal hyperalgesia. These findings indicate that SP-induced thermal hyperalgesia may be maintained through the enhanced responsiveness of NMDA or AMPA receptors, but not the receptor of SP, mediated by kinases.

  2. LTP requires a reserve pool of glutamate receptors independent of subunit type. (United States)

    Granger, Adam J; Shi, Yun; Lu, Wei; Cerpas, Manuel; Nicoll, Roger A


    Long-term potentiation (LTP) of synaptic transmission is thought to be an important cellular mechanism underlying memory formation. A widely accepted model posits that LTP requires the cytoplasmic carboxyl tail (C-tail) of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GluA1. To find the minimum necessary requirement of the GluA1 C-tail for LTP in mouse CA1 hippocampal pyramidal neurons, we used a single-cell molecular replacement strategy to replace all endogenous AMPA receptors with transfected subunits. In contrast to the prevailing model, we found no requirement of the GluA1 C-tail for LTP. In fact, replacement with the GluA2 subunit showed normal LTP, as did an artificially expressed kainate receptor not normally found at these synapses. The only conditions under which LTP was impaired were those with markedly decreased AMPA receptor surface expression, indicating a requirement for a reserve pool of receptors. These results demonstrate the synapse's remarkable flexibility to potentiate with a variety of glutamate receptor subtypes, requiring a fundamental change in our thinking with regard to the core molecular events underlying synaptic plasticity.

  3. Mechanisms for Antagonistic Regulation of AMPA and NMDA-D1 Receptor Complexes at Postsynaptic Sites (United States)

    Schumann, Johann; Scheler, Gabriele


    From the analysis of these pathways we conclude that postsynaptic processes that regulate synaptic transmission undergo significant cross-talk with respect to glutamatergic and neuromodulatory (dopamine) signals. The main hypothesis is that of a compensatory regulation, a competitive switch between the induction of increased AMPA conductance by CaMKII-dependent phosphorylation and reduced expression of PP2A, and increased D1 receptor sensitivity and expression by increased PKA, PP2A and decreased PP-1/calcineurin expression. Both types of plasticity are induced by NMDA receptor activation and increased internal calcium, they require different internal conditions to become expressed. Specifically we propose that AMPA regulation and D1 regulation are inversely coupled;The net result may be a bifurcation of synaptic state into predominantly AMPA or NMDA-D1 synapses. This could have functional consequences: stable connections for AMPA and conditional gating for NMDA-D1 synapses.

  4. Contextual learning requires synaptic AMPA receptor delivery in the hippocampus. (United States)

    Mitsushima, Dai; Ishihara, Kouji; Sano, Akane; Kessels, Helmut W; Takahashi, Takuya


    The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. By combining viral-mediated in vivo gene delivery with in vitro patch-clamp recordings, we found that the inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, delivered GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus. To block the synaptic delivery of endogenous AMPARs, we expressed a fragment of the GluR1-cytoplasmic tail (the 14-aa GluR1 membrane-proximal region with two serines mutated to phospho-mimicking aspartates: MPR-DD). MPR-DD prevented learning-driven synaptic AMPAR delivery in CA1 neurons. Bilateral expression of MPR-DD in the CA1 region of the rat impaired inhibitory avoidance learning, indicating that synaptic GluR1 trafficking in the CA1 region of the hippocampus is required for encoding contextual fear memories. The fraction of CA1 neurons that underwent synaptic strengthening positively correlated with the performance in the inhibitory avoidance fear memory task. These data suggest that the robustness of a contextual memory depends on the number of hippocampal neurons that participate in the encoding of a memory trace.

  5. 1,2,3-triazolyl amino acids as AMPA receptor ligands

    DEFF Research Database (Denmark)

    Stanley, Nathan J.; Pedersen, Daniel Sejer; Nielsen, Birgitte


    The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further...

  6. PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking. (United States)

    Lu, Wei; Ziff, Edward B


    PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.

  7. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Anubhuti Goel

    Full Text Available Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+-permeable AMPA receptors (CP-AMPARs. However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1 subunit at the serine 845 (S845 site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants, which is a substrate of cAMP-dependent kinase (PKA, show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  8. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity. (United States)

    Goel, Anubhuti; Xu, Linda W; Snyder, Kevin P; Song, Lihua; Goenaga-Vazquez, Yamila; Megill, Andrea; Takamiya, Kogo; Huganir, Richard L; Lee, Hey-Kyoung


    Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  9. Natural reward experience alters AMPA and NMDA receptor distribution and function in the nucleus accumbens.

    Directory of Open Access Journals (Sweden)

    Kyle K Pitchers

    Full Text Available Natural reward and drugs of abuse converge upon the mesolimbic system which mediates motivation and reward behaviors. Drugs induce neural adaptations in this system, including transcriptional, morphological, and synaptic changes, which contribute to the development and expression of drug-related memories and addiction. Previously, it has been reported that sexual experience in male rats, a natural reward behavior, induces similar neuroplasticity in the mesolimbic system and affects natural reward and drug-related behavior. The current study determined whether sexual experience causes long-lasting changes in mating, or ionotropic glutamate receptor trafficking or function in the nucleus accumbens (NAc, following 3 different reward abstinence periods: 1 day, 1 week, or 1 month after final mating session. Male Sprague Dawley rats mated during 5 consecutive days (sexual experience or remained sexually naïve to serve as controls. Sexually experienced males displayed facilitation of initiation and performance of mating at each time point. Next, intracellular and membrane surface expression of N-methyl-D-aspartate (NMDA: NR1 subunit and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA: GluA1, GluA2 subunits receptors in the NAc was determined using a bis(sulfosuccinimidylsuberate (BS(3 protein cross-linking assay followed by Western Blot analysis. NR1 expression was increased at 1 day abstinence both at surface and intracellular, but decreased at surface at 1 week of abstinence. GluA2 was increased intracellularly at 1 week and increased at the surface after 1 month of abstinence. Finally, whole-cell patch clamp electrophysiological recordings determined reduced AMPA/NMDA ratio of synaptic currents in NAc shell neurons following stimulation of cortical afferents in sexually experienced males after all reward abstinence periods. Together, these data show that sexual experience causes long-term alterations in glutamate receptor expression and

  10. Induction of Increased Intraceilular Calcium in Astrocytes by Glutamate through Activating NMDA and AMPA Receptors

    Institute of Scientific and Technical Information of China (English)

    张蕲; 胡波; 孙圣刚; 童萼塘


    To study the effect of glutamate on the intracellular calcium signal of pure cultured ratastrocytes and the role of NMDA and AMPA receptors in the procedure, the change of calcium sig-nal was investigated by monitoring the fluctuation of intracellular Ca2+ concentration ([Ca2+]i) onthe basis of Fura-2 single cell fluorescent ratio (F345/F380). The changes in the effect of glutamateon the intracellular calcium signal were observed after blockage of NMDA and(or) AMPA recep-tors. It was found that L-glutamate could induce an increased [Ca2+]i in most of the cells in concen-tration- and time-dependent manner. D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5, a selec-tive antagonist of the NMDA receptor) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selec-tive antagonist of the AMPA receptor) could abolish the effects of NMDA and AMPA respectively.Th.e treatment of D-AP-5 and CNQX simultaneously or respectively could attenuate the effect of L-glutamate at varying degrees. All these indicated that glutamate could modulate intracellular Ca2+of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA re-ceptors took part in the complex mechanisms.

  11. NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes

    Institute of Scientific and Technical Information of China (English)

    Bo HU; Sheng-gang SUN; E-tang TONG


    AIM: To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors in the procedure. METHODS: The fluorescence of calcium was measured by Fura-2/AM (F345/F380).RESULTS: L-Glutamate induced [Ca2+]i increase in most of the cells in concentration- and time-dependent manner.NMDA 50 mmol/L induced the fluorescence increase by almost three to four times, while the effect of AMPA 50mmol/L was just half of that of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5; a selective antagonist of the NMDA receptor). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor)abolished the effects of NMDA and AMPA, respectively. D-AP-5 and CNQX simultaneously or respectively attenuated the effect of L-glutamate at different degrees, but could not abolish it entirely. CONCLUSION: Glutamate modulated intracellular Ca2+ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.

  12. mTOR Is Essential for Corticosteroid Effects on Hippocampal AMPA Receptor Function and Fear Memory (United States)

    Xiong, Hui; Casse, Frédéric; Zhou, Yang; Zhou, Ming; Xiong, Zhi-Qi; Joëls, Marian; Martin, Stéphane; Krugers, Harm J.


    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptors (AMPARs), which are crucial for synaptic plasticity and memory formation. Combining a live imaging fluorescent recovery after photobleaching approach…

  13. Dual-specific Phosphatase-6 (Dusp6) and ERK Mediate AMPA Receptor-induced Oligodendrocyte Death* (United States)

    Domercq, Maria; Alberdi, Elena; Sánchez-Gómez, Maria Victoria; Ariz, Usue; Pérez-Samartín, Alberto; Matute, Carlos


    Oligodendrocytes, the myelinating cells of the CNS, are highly vulnerable to glutamate excitotoxicity, a mechanism involved in tissue damage in multiple sclerosis. Thus, understanding oligodendrocyte death at the molecular level is important to develop new therapeutic approaches to treat the disease. Here, using microarray analysis and quantitative PCR, we observed that dual-specific phosphatase-6 (Dusp6), an extracellular regulated kinase-specific phosphatase, is up-regulated in oligodendrocyte cultures as well as in optic nerves after AMPA receptor activation. In turn, Dusp6 is overexpressed in optic nerves from multiple sclerosis patients before the appearance of evident damage in this structure. We further analyzed the role of Dusp6 and ERK signaling in excitotoxic oligodendrocyte death and observed that AMPA receptor activation induces a rapid increase in ERK1/2 phosphorylation. Blocking Dusp6 expression, which enhances ERK1/2 phosphorylation, significantly diminished AMPA receptor-induced oligodendrocyte death. In contrast, MAPK/ERK pathway inhibition with UO126 significantly potentiates excitotoxic oligodendrocyte death and increases cytochrome c release, mitochondrial depolarization, and mitochondrial calcium overload produced by AMPA receptor stimulation. Upstream analysis demonstrated that MAPK/ERK signaling alters AMPA receptor properties. Indeed, Dusp6 overexpression as well as incubation with UO126 produced an increase in AMPA receptor-induced inward currents and cytosolic calcium overload. Together, these data suggest that levels of phosphorylated ERK, controlled by Dusp6 phosphatase, regulate glutamate receptor permeability and oligodendroglial excitotoxicity. Therefore, targeting Dusp6 may be a useful strategy to prevent oligodendrocyte death in multiple sclerosis and other diseases involving CNS white matter. PMID:21300799

  14. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-containing NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Li-Jun Li


    Full Text Available NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs, but not GluN2B-containing NMDA receptors (GluN2BRs, to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation.

  15. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors (United States)

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi


    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation.

  16. Synthesis and enantiopharmacology of new AMPA-kainate receptor agonists

    DEFF Research Database (Denmark)

    Conti, P; De Amici, M; De Sarro, G


    . The convulsant properties of all the compounds were evaluated in vivo on DBA/2 mice after icv injection. CIP-A showed a convulsant activity, measured as tonus and clonus seizures, 18-65 times higher than that produced by AMPA. It was also quite active after ip administration, since it induced seizures in mice...

  17. Requirement of AMPA receptor stimulation for the sustained antidepressant activity of ketamine and LY341495 during the forced swim test in rats. (United States)

    Koike, Hiroyuki; Chaki, Shigeyuki


    Ketamine, a non-competitive N-methyl-d-aspartate receptor antagonist, and group II metabotropic glutamate (mGlu2/3) receptor antagonists produce antidepressant effects in animal models of depression, which last for at least 24h, through the transient increase in glutamate release, leading to activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor. Both ketamine and an mGlu2/3 receptor antagonist reportedly increase the expression of GluR1, an AMPA receptor subunit, within 24h, which may account for the sustained enhancement of excitatory synaptic transmission following ketamine administration. However, whether the sustained increase in AMPA receptor-mediated synaptic transmission is associated with the antidepressant effects of ketamine and mGlu2/3 receptor antagonists has not yet been investigated. In the present study, to address this question, we tested whether AMPA receptor stimulation at 24h after a single injection of ketamine or an mGlu2/3 receptor antagonist, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY341495) was necessary for the antidepressant effect of these compounds using a forced swim test in rats. A single injection of ketamine or LY341495 at 24h before the test significantly decreased the immobility time. An AMPA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), administered 30min prior to the test significantly and dose-dependently reversed the antidepressant effects of ketamine and LY341495, while NBQX itself had no effect on the immobility time. Our findings suggest that AMPA receptor stimulation at 24h after a single injection of ketamine or LY341495 is required to produce the anti-immobility effects of these compounds. Moreover, the present results provide additional evidence that an mGlu2/3 receptor antagonist may share some of neural mechanisms with ketamine to exert antidepressant effects.

  18. Shisa6 traps AMPA receptors at postsynaptic sites and prevents their desensitization during synaptic activity. (United States)

    Klaassen, Remco V; Stroeder, Jasper; Coussen, Françoise; Hafner, Anne-Sophie; Petersen, Jennifer D; Renancio, Cedric; Schmitz, Leanne J M; Normand, Elisabeth; Lodder, Johannes C; Rotaru, Diana C; Rao-Ruiz, Priyanka; Spijker, Sabine; Mansvelder, Huibert D; Choquet, Daniel; Smit, August B


    Trafficking and biophysical properties of AMPA receptors (AMPARs) in the brain depend on interactions with associated proteins. We identify Shisa6, a single transmembrane protein, as a stable and directly interacting bona fide AMPAR auxiliary subunit. Shisa6 is enriched at hippocampal postsynaptic membranes and co-localizes with AMPARs. The Shisa6 C-terminus harbours a PDZ domain ligand that binds to PSD-95, constraining mobility of AMPARs in the plasma membrane and confining them to postsynaptic densities. Shisa6 expressed in HEK293 cells alters GluA1- and GluA2-mediated currents by prolonging decay times and decreasing the extent of AMPAR desensitization, while slowing the rate of recovery from desensitization. Using gene deletion, we show that Shisa6 increases rise and decay times of hippocampal CA1 miniature excitatory postsynaptic currents (mEPSCs). Shisa6-containing AMPARs show prominent sustained currents, indicating protection from full desensitization. Accordingly, Shisa6 prevents synaptically trapped AMPARs from depression at high-frequency synaptic transmission.

  19. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory (United States)

    Cazakoff, Brittany N.; Howland, John G.


    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  20. Studies on Aryl-Substituted Phenylalanines: Synthesis, Activity, and Different Binding Modes at AMPA Receptors

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Frydenvang, Karla Andrea; Pickering, Darryl S;


    A series of racemic aryl-substituted phenylalanines was synthesized and evaluated in vitro at recombinant rat GluA1−3, at GluK1−3, and at native AMPA receptors. The individual enantiomers of two target compounds, (RS)-2-amino-3-(3,4-dichloro-5-(5-hydroxypyridin-3-yl)phenyl)- propanoic acid (37...

  1. GABA B receptor subunit expression in glia. (United States)

    Charles, K J; Deuchars, J; Davies, C H; Pangalos, M N


    GABA(B) receptor subunits are widely expressed on neurons throughout the CNS, at both pre- and postsynaptic sites, where they mediate the late, slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. The existence of functional GABA(B) receptors on nonneuronal cells has been reported previously, although the molecular composition of these receptors has not yet been described. Here we demonstrate for the first time, using immunohistochemistry the expression of GABA(B1a), GABA(B1b), and GABA(B2) on nonneuronal cells of the rat CNS. All three principle GABA(B) receptor subunits were expressed on these cells irrespective of whether they had been cultured or found within brain tissue sections. At the ultrastructural level GABA(B) receptor subunits were expressed on astrocytic processes surrounding both symmetrical and assymetrical synapses in the CA1 subregion of the hippocampus. In addition, GABA(B1a), GABA(B1b), and GABA(B2) receptor subunits were expressed on activated microglia in culture but were not found on myelin forming oligodendrocytes in the white matter of rat spinal cord. Together these data demonstrate that the obligate subunits of functional GABA(B) receptors are expressed in astrocytes and microglia in the rat CNS.

  2. Acoustic trauma slows AMPA receptor‐mediated EPSCs in the auditory brainstem, reducing GluA4 subunit expression as a mechanism to rescue binaural function (United States)

    Pilati, Nadia; Linley, Deborah M.; Selvaskandan, Haresh; Uchitel, Osvaldo; Hennig, Matthias H.; Kopp‐Scheinpflug, Cornelia


    Key points Lateral superior olive (LSO) principal neurons receive AMPA receptor (AMPAR) ‐ and NMDA receptor (NMDAR)‐mediated EPSCs and glycinergic IPSCs.Both EPSCs and IPSCs have slow kinetics in prehearing animals, which during developmental maturation accelerate to sub‐millisecond decay time‐constants. This correlates with a change in glutamate and glycine receptor subunit composition quantified via mRNA levels.The NMDAR‐EPSCs accelerate over development to achieve decay time‐constants of 2.5 ms. This is the fastest NMDAR‐mediated EPSC reported.Acoustic trauma (AT, loud sounds) slow AMPAR‐EPSC decay times, increasing GluA1 and decreasing GluA4 mRNA.Modelling of interaural intensity difference suggests that the increased EPSC duration after AT shifts interaural level difference to the right and compensates for hearing loss.Two months after AT the EPSC decay times recovered to control values.Synaptic transmission in the LSO matures by postnatal day 20, with EPSCs and IPSCs having fast kinetics. AT changes the AMPAR subunits expressed and slows the EPSC time‐course at synapses in the central auditory system. Abstract Damaging levels of sound (acoustic trauma, AT) diminish peripheral synapses, but what is the impact on the central auditory pathway? Developmental maturation of synaptic function and hearing were characterized in the mouse lateral superior olive (LSO) from postnatal day 7 (P7) to P96 using voltage‐clamp and auditory brainstem responses. IPSCs and EPSCs show rapid acceleration during development, so that decay kinetics converge to similar sub‐millisecond time‐constants (τ, 0.87 ± 0.11 and 0.77 ± 0.08 ms, respectively) in adult mice. This correlated with LSO mRNA levels for glycinergic and glutamatergic ionotropic receptor subunits, confirming a switch from Glyα2 to Glyα1 for IPSCs and increased expression of GluA3 and GluA4 subunits for EPSCs. The NMDA receptor (NMDAR)‐EPSC decay τ accelerated from >40 ms in

  3. Opposite modulation of brain stimulation reward by NMDA and AMPA receptors in the ventral tegmental area.

    Directory of Open Access Journals (Sweden)

    Charles eDucrot


    Full Text Available Previous studies have shown that blockade of ventral midbrain (VM glutamate N-Methyl-D-Aspartate (NMDA receptors induces reward, stimulates forward locomotion and enhances brain stimulation reward. Glutamate induces two types of excitatory response on VM neurons, a fast and short lasting depolarisation mediated by a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA receptors and a longer lasting depolarization mediated by NMDA receptors. A role for the two glutamate receptors in modulation of VM neuronal activity is evidenced by the functional change in AMPA and NMDA synaptic responses that result from repeated exposure to reward. Since both receptors contribute to the action of glutamate on VM neuronal activity, we studied the effects of VM AMPA and NMDA receptor blockade on reward induced by electrical brain stimulation. Experiments were performed on rats trained to self-administer electrical pulses in the medial posterior mesencephalon. Reward thresholds were measured with the curve-shift paradigm before and for two hours after bilateral VM microinjections of the AMPA antagonist, NBQX (2,3,-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(fquinoxaline-7-sulfonamide, 0, 80, and 800 pmol/0.5ul/side and of a single dose (0.825 nmol/0.5ul/side of the NMDA antagonist, PPPA (2R,4S-4-(3-Phosphonopropyl-2-piperidinecarboxylic acid. NBQX produced a dose-dependent increase in reward threshold with no significant change in maximum rate of responding. Whereas PPPA injected at the same VM sites produced a significant time dependent decrease in reward threshold and increase in maximum rate of responding. We found a negative correlation between the magnitude of the attenuation effect of NBQX and the enhancement effect of PPPA; moreover, NBQX and PPPA were most effective when injected respectively into the anterior and posterior VM. These results suggest that glutamate acts on different receptor sub-types, most likely located on different VM neurons, to modulate

  4. Opposite modulation of brain stimulation reward by NMDA and AMPA receptors in the ventral tegmental area. (United States)

    Ducrot, Charles; Fortier, Emmanuel; Bouchard, Claude; Rompré, Pierre-Paul


    Previous studies have shown that blockade of ventral tegmental area (VTA) glutamate N-Methyl-D-Aspartate (NMDA) receptors induces reward, stimulates forward locomotion and enhances brain stimulation reward. Glutamate induces two types of excitatory response on VTA neurons, a fast and short lasting depolarization mediated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors and a longer lasting depolarization mediated by NMDA receptors. A role for the two glutamate receptors in modulation of VTA neuronal activity is evidenced by the functional change in AMPA and NMDA synaptic responses that result from repeated exposure to reward. Since both receptors contribute to the action of glutamate on VTA neuronal activity, we studied the effects of VTA AMPA and NMDA receptor blockade on reward induced by electrical brain stimulation. Experiments were performed on rats trained to self-administer electrical pulses in the medial posterior mesencephalon. Reward thresholds were measured with the curve-shift paradigm before and for 2 h after bilateral VTA microinjections of the AMPA antagonist, NBQX (2,3,-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide, 0, 80, and 800 pmol/0.5 μl/side) and of a single dose (0.825 nmol/0.5 μl/side) of the NMDA antagonist, PPPA (2R,4S)-4-(3-Phosphonopropyl)-2-piperidinecarboxylic acid). NBQX produced a dose-dependent increase in reward threshold with no significant change in maximum rate of responding. Whereas PPPA injected at the same VTA sites produced a significant time dependent decrease in reward threshold and increase in maximum rate of responding. We found a negative correlation between the magnitude of the attenuation effect of NBQX and the enhancement effect of PPPA; moreover, NBQX and PPPA were most effective when injected, respectively, into the anterior and posterior VTA. These results suggest that glutamate acts on different receptor sub-types, most likely located on different VTA neurons, to

  5. in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives. (United States)

    Azam, Faizul; Abugrain, Ismaiel Mohamed; Sanalla, Mohamed Hussin; Elnaas, Radwan Fatahalla; Rajab, Ibrahim Abdassalam Ibn


    Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders.

  6. Computational study of the evolutionary relationships of the ionotropic receptors NMDA, AMPA and kainate in four species of primates.


    Moreno-Pedraza, Francy Johanna; Grupo de Bioquímica Molecular Computacional y Bioinformática, Departamento de Bioquímica, Facultad de Ciencias. Pontificia Universidad Javeriana, Carrera 7 No. 43-82 Ed. 52, Bogotá,; Lareo, Leonardo René; Grupo de Bioquímica Molecular Computacional y Bioinformática, Departamento de Bioquímica, Facultad de Ciencias. Pontificia Universidad Javeriana, Carrera 7 No. 43-82 Ed. 52, Bogotá,; Reyes-Montaño, Edgar Antonio; Grupo de Investigación en Proteínas (GRIP) Departamento de Química, Universidad Nacional de Colombia, Ciudad Universitaria, Edificio 451, Bogotá


    Objective. To identify the influence of changes on the secondary structure and evolutionary relationship of NMDA, AMPA and kainate receptors in Homo sapiens, Pan troglodytes, Pongo pygmaeus and Macaca mulatta. Materials and methods. We identified 91 sequences for NMDA, AMPA and kainate receptors and analyzed with software for predicting secondary structure, phosphorylation sites, multiple alignments, selection of protein evolution models and phylogenetic prediction. Results. We found that s...

  7. Hippocampal GluA1-containing AMPA receptors mediate context-dependent sensitization to morphine. (United States)

    Xia, Yan; Portugal, George S; Fakira, Amanda K; Melyan, Zara; Neve, Rachael; Lee, H Thomas; Russo, Scott J; Liu, Jie; Morón, Jose A


    Glutamatergic systems, including AMPA receptors (AMPARs), are involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are not fully understood. In the present study, we investigated the effects of repeated morphine administration on AMPAR expression, synaptic plasticity, and context-dependent behavioral sensitization to morphine. We found that morphine treatment produced changes of synaptic AMPAR expression in the hippocampus, a brain area that is critically involved in learning and memory. These changes could be observed 1 week after the treatment, but only when mice developed context-dependent behavioral sensitization to morphine in which morphine treatment was associated with drug administration environment. Context-dependent behavioral sensitization to morphine was also associated with increased basal synaptic transmission and disrupted hippocampal long-term potentiation (LTP), whereas these effects were less robust when morphine administration was not paired with the drug administration environment. Interestingly, some effects may be related to the prior history of morphine exposure in the drug-associated environment, since alterations of AMPAR expression, basal synaptic transmission, and LTP were observed in mice that received a saline challenge 1 week after discontinuation of morphine treatment. Furthermore, we demonstrated that phosphorylation of GluA1 AMPAR subunit plays a critical role in the acquisition and expression of context-dependent behavioral sensitization, as this behavior is blocked by a viral vector that disrupts GluA1 phosphorylation. These data provide evidence that glutamatergic signaling in the hippocampus plays an important role in context-dependent sensitization to morphine and supports further investigation of glutamate-based strategies for treating opiate addiction.

  8. Drug-driven AMPA receptor redistribution mimicked by selective dopamine neuron stimulation.

    Directory of Open Access Journals (Sweden)

    Matthew T C Brown

    Full Text Available BACKGROUND: Addictive drugs have in common that they cause surges in dopamine (DA concentration in the mesolimbic reward system and elicit synaptic plasticity in DA neurons of the ventral tegmental area (VTA. Cocaine for example drives insertion of GluA2-lacking AMPA receptors (AMPARs at glutamatergic synapes in DA neurons. However it remains elusive which molecular target of cocaine drives such AMPAR redistribution and whether other addictive drugs (morphine and nicotine cause similar changes through their effects on the mesolimbic DA system. METHODOLOGY/PRINCIPAL FINDINGS: We used in vitro electrophysiological techniques in wild-type and transgenic mice to observe the modulation of excitatory inputs onto DA neurons by addictive drugs. To observe AMPAR redistribution, post-embedding immunohistochemistry for GluA2 AMPAR subunit was combined with electron microscopy. We also used a double-floxed AAV virus expressing channelrhodopsin together with a DAT Cre mouse line to selectively express ChR2 in VTA DA neurons. We find that in mice where the effect of cocaine on the dopamine transporter (DAT is specifically blocked, AMPAR redistribution was absent following administration of the drug. Furthermore, addictive drugs known to increase dopamine levels cause a similar AMPAR redistribution. Finally, activating DA VTA neurons optogenetically is sufficient to drive insertion of GluA2-lacking AMPARs, mimicking the changes observed after a single injection of morphine, nicotine or cocaine. CONCLUSIONS/SIGNIFICANCE: We propose the mesolimbic dopamine system as a point of convergence at which addictive drugs can alter neural circuits. We also show that direct activation of DA neurons is sufficient to drive AMPAR redistribution, which may be a mechanism associated with early steps of non-substance related addictions.

  9. Bi-directional modulation of AMPA receptor unitary conductance by synaptic activity

    Directory of Open Access Journals (Sweden)

    Matthews Paul


    Full Text Available Abstract Background Knowledge of how synapses alter their efficiency of communication is central to the understanding of learning and memory. The most extensively studied forms of synaptic plasticity are long-term potentiation (LTP and its counterpart long-term depression (LTD of AMPA receptor-mediated synaptic transmission. In the CA1 region of the hippocampus, it has been shown that LTP often involves a rapid increase in the unitary conductance of AMPA receptor channels. However, LTP can also occur in the absence of any alteration in AMPA receptor unitary conductance. In the present study we have used whole-cell dendritic recording, failures analysis and non-stationary fluctuation analysis to investigate the mechanism of depotentiation of LTP. Results We find that when LTP involves an increase in unitary conductance, subsequent depotentiation invariably involves the return of unitary conductance to pre-LTP values. In contrast, when LTP does not involve a change in unitary conductance then depotentiation also occurs in the absence of any change in unitary conductance, indicating a reduction in the number of activated receptors as the most likely mechanism. Conclusions These data show that unitary conductance can be bi-directionally modified by synaptic activity. Furthermore, there are at least two distinct mechanisms to restore synaptic strength from a potentiated state, which depend upon the mechanism of the previous potentiation.

  10. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells


    Yu LIU; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June


    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca2+ and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the durati...

  11. AMPA Receptor-Induced Local Brain-Derived Neurotrophic Factor Signaling Mediates Motor Recovery after Stroke


    Clarkson, Andrew N.; Overman, Justine J; Zhong, Sheng; Mueller, Rudolf; Lynch, Gary; Carmichael, S. Thomas


    Stroke is the leading cause of adult disability. Recovery after stroke shares similar molecular and cellular properties with learning and memory. A main component of learning-induced plasticity involves signaling through AMPA receptors (AMPARs). We systematically tested the role of AMPAR function in motor recovery in a mouse model of focal stroke. AMPAR function controls functional recovery beginning 5 d after the stroke. Positive allosteric modulators of AMPARs enhance recovery of limb contr...

  12. X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism. (United States)

    Chen, Lei; Dürr, Katharina L; Gouaux, Eric


    AMPA-sensitive glutamate receptors are crucial to the structural and dynamic properties of the brain, to the development and function of the central nervous system, and to the treatment of neurological conditions from depression to cognitive impairment. However, the molecular principles underlying AMPA receptor activation have remained elusive. We determined multiple x-ray crystal structures of the GluA2 AMPA receptor in complex with a Conus striatus cone snail toxin, a positive allosteric modulator, and orthosteric agonists, at 3.8 to 4.1 angstrom resolution. We show how the toxin acts like a straightjacket on the ligand-binding domain (LBD) "gating ring," restraining the domains via both intra- and interdimer cross-links such that agonist-induced closure of the LBD "clamshells" is transduced into an irislike expansion of the gating ring. By structural analysis of activation-enhancing mutants, we show how the expansion of the LBD gating ring results in pulling forces on the M3 helices that, in turn, are coupled to ion channel gating.

  13. Precision therapy for a new disorder of AMPA receptor recycling due to mutations in ATAD1 (United States)

    Ahrens-Nicklas, Rebecca C.; Umanah, George K.E.; Sondheimer, Neal; Deardorff, Matthew A.; Wilkens, Alisha B.; Conlin, Laura K.; Santani, Avni B.; Nesbitt, Addie; Juulsola, Jane; Ma, Erica; Dawson, Ted M.; Dawson, Valina L.


    Objective: ATAD1 encodes Thorase, a mediator of α-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) receptor recycling; in this work, we characterized the phenotype resulting from ATAD1 mutations and developed a targeted therapy in both mice and humans. Methods: Using exome sequencing, we identified a novel ATAD1 mutation (p.E276X) as the etiology of a devastating neurologic disorder characterized by hypertonia, seizures, and death in a consanguineous family. We postulated that pathogenesis was a result of excessive AMPA receptor activity and designed a targeted therapeutic approach using perampanel, an AMPA-receptor antagonist. Results: Perampanel therapy in ATAD1 knockout mice reversed behavioral defects, normalized brain MRI abnormalities, prevented seizures, and prolonged survival. The ATAD1 patients treated with perampanel showed improvement in hypertonicity and resolution of seizures. Conclusions: This work demonstrates that identification of novel monogenic neurologic disorders and observation of response to targeted therapeutics can provide important insights into human nervous system functioning. PMID:28180185

  14. Oligomeric amyloid-{beta} inhibits the proteolytic conversion of brain-derived neurotrophic factor (BDNF), AMPA receptor trafficking, and classical conditioning. (United States)

    Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce


    Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

  15. Role of AMPA and NMDA receptors and back-propagating action potentials in spike timing-dependent plasticity. (United States)

    Fuenzalida, Marco; Fernández de Sevilla, David; Couve, Alejandro; Buño, Washington


    The cellular mechanisms that mediate spike timing-dependent plasticity (STDP) are largely unknown. We studied in vitro in CA1 pyramidal neurons the contribution of AMPA and N-methyl-d-aspartate (NMDA) components of Schaffer collateral (SC) excitatory postsynaptic potentials (EPSPs; EPSP(AMPA) and EPSP(NMDA)) and of the back-propagating action potential (BAP) to the long-term potentiation (LTP) induced by a STDP protocol that consisted in pairing an EPSP and a BAP. Transient blockade of EPSP(AMPA) with 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX) during the STDP protocol prevented LTP. Contrastingly LTP was induced under transient inhibition of EPSP(AMPA) by combining SC stimulation, an imposed EPSP(AMPA)-like depolarization, and BAP or by coupling the EPSP(NMDA) evoked under sustained depolarization (approximately -40 mV) and BAP. In Mg(2+)-free solution EPSP(NMDA) and BAP also produced LTP. Suppression of EPSP(NMDA) or BAP always prevented LTP. Thus activation of NMDA receptors and BAPs are needed but not sufficient because AMPA receptor activation is also obligatory for STDP. However, a transient depolarization of another origin that unblocks NMDA receptors and a BAP may also trigger LTP.

  16. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. (United States)

    Ahmed, Ahmed H; Oswald, Robert E


    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  17. An AMPA receptor potentiator modulates hippocampal expression of BDNF: an in vivo study. (United States)

    Mackowiak, Marzena; O'Neill, Michael J; Hicks, Caroline A; Bleakman, David; Skolnick, Phil


    AMPA receptor activation has been demonstrated to increase the neuronal expression of brain derived neurotrophic factor (BDNF). In the present study, we investigated the effect of a novel AMPA receptor potentiator (LY404187) and its active isomer (LY451646) on the expression of BDNF protein and mRNA, as well as TrkB mRNA in rat hippocampus. LY404187 administered for 7 days (1 mg/kg) significantly increased the number of BDNF immunopositive cells in the dentate gyrus, but not other hippocampal subfields. Chronic treatment (7 days) with LY451646 (0.5 mg/kg, comparable to 1 mg/kg of LY404187) increased the level of both BDNF and TrkB mRNA expression in the dentate gyrus, CA3 and CA4 of the hippocampus. However, chronic treatment with lower doses of LY451646 (0.125 and 0.25 mg/kg) decreased the level of BDNF and TrkB mRNA in hippocampus, whilst the highest used dose of LY451646 (1 mg/kg) had no effect on BDNF and TrkB mRNA in hippocampus. In contrast, acute treatment with LY451646 produced an increase in BDNF mRNA levels at doses of 0.125 and 0.25 mg/kg in the hippocampus (CA4, CA3 and dentate gyrus, but not in CA1). LY451646 at 0.5 mg/kg had no effect, but at 1.0 mg/kg decreased the level of BDNF mRNA in hippocampus. Acute treatment with LY451646 did not affect the TrkB receptor mRNA levels in hippocampus. Our results demonstrate that biarylpropylsulfonamide AMPA receptor potentiators are capable of modulating the expression of BDNF and TrkB mRNA in a dose- and time-dependent manner. The increase in both BDNF protein and mRNA expression in the dentate gyrus but not in CA1 indicates a specific role of AMPA receptors in the regulation of BDNF expression in this hippocampal subfield. The regulation of BDNF expression by biarylpropylsulfonamids such as LY451646 may have important therapeutical implications for this class of molecule in the treatment of depression and other CNS disorders.

  18. Shape-induced asymmetric diffusion in dendritic spines allows efficient synaptic AMPA receptor trapping. (United States)

    Kusters, Remy; Kapitein, Lukas C; Hoogenraad, Casper C; Storm, Cornelis


    Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.

  19. (S)-homo-AMPA, a specific agonist at the mGlu6 subtype of metabotropic glutamic acid receptors

    DEFF Research Database (Denmark)

    Ahmadian, H; Nielsen, B; Bräuner-Osborne, Hans;


    of the spectroscopic configurational assignments. The activities of 6 and 7 at ionotropic EAA (iGlu) receptors and at mGlu1-7 were studied. (S)-Homo-AMPA (6) was shown to be a specific agonist at mGlu6 (EC50 = 58 +/- 11 microM) comparable in potency with the endogenous mGlu agonist (S)-glutamic acid (EC50 = 20 +/- 3......Our previous publication (J. Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the mGlu6 subtype of metabotropic excitatory amino acid (EAA) receptors. Homo-AMPA has already become a standard agonist...... microM). Although Homo-AMPA did not show significant effects at iGlu receptors, (R)-Homo-AMPA (7), which was inactive at mGlu1-7, turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist (IC50 = 131 +/- 18 microM)....

  20. AMPA receptor modulators have different impact on hippocampal pyramidal cells and interneurons. (United States)

    Xia, Y-F; Arai, A C


    Positive modulators of AMPA receptors enhance synaptic plasticity and memory encoding. Facilitation of AMPA receptor currents not only results in enhanced activation of excitatory neurons but also increases the activity of inhibitory interneurons by up-modulating their excitatory input. However, little is known about the effects of these modulators on cells other than pyramidal neurons and about their impact on local microcircuits. This study examined the effects of members from three subfamilies of modulators (mainly CX516, CX546 and cyclothiazide) on excitatory synaptic responses in four classes of hippocampal CA1 neurons and on excitatory and disynaptically induced inhibitory field potentials in hippocampal slices. Effects on excitatory postsynaptic currents (EPSCs) were examined in pyramidal cells, in two types of inhibitory interneurons located in stratum radiatum and oriens, and in stratum radiatum giant cells, a novel type of excitatory neuron. With CX516, increases in EPSC amplitude in pyramidal cells were two to three times larger than in interneurons and six times larger than in radiatum giant cells. The effects of CX546 on response duration similarly were largest in pyramidal cells. However, this drug also strongly differentiated between stratum oriens and radiatum interneurons with increases being four times larger in the latter. In contrast, cyclothiazide had similar effects on response duration in all cell types. In field recordings, CX516 was several times more potent in enhancing excitatory postsynaptic potentials (EPSPs) than feedback or feedforward circuits, as expected from its larger influence on pyramidal cells. In contrast, BDP-20, a CX546 analog, was more potent in enhancing feedforward inhibition than either EPSPs or feedback inhibition. This preference for feedforward over feedback circuits is probably related to its higher potency in stratum radiatum versus oriens interneurons. Taken together, AMPA receptor modulators differ substantially

  1. AMPA and GABA receptor antagonists and their interaction in rats with a genetic form of absence epilepsy

    NARCIS (Netherlands)

    Kaminski, R.M.; Rijn, C.M. van; Turski, W.A.; Czuczwar, S.J.; Luijtelaar, E.L.J.M. van


    The effects of combined and single administration of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, 7,8-methylenedioxy-1-(4-aminophenyl)-4-methyl-3-acetyl-4,5-dihydro-2,3 -benzodiazepine (LY 300164), and of the GABAB receptor antagonist -aminopropyl-n-butyl-phosp

  2. Competitive antagonism of AMPA receptors by ligands of different classes

    DEFF Research Database (Denmark)

    Hogner, Anders; Greenwood, Jeremy R; Liljefors, Tommy;


    that ATPO and DNQX stabilize an open form of the ligand-binding core by different sets of interactions. Computational techniques are used to quantify the differences between these two ligands and to map the binding site. The isoxazole moiety of ATPO acts primarily as a spacer, and other scaffolds could......-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO) in complex with the ligand-binding core of the receptor. Comparison with the only previous structure of the ligand-binding core in complex with an antagonist, 6,7-dinitro-2,3-quinoxalinedione (DNQX) (Armstrong, N.; Gouaux, E. Neuron 2000, 28, 165-181), reveals...

  3. Restoring Light Sensitivity in Blind Retinae Using a Photochromic AMPA Receptor Agonist. (United States)

    Laprell, L; Hüll, K; Stawski, P; Schön, C; Michalakis, S; Biel, M; Sumser, M P; Trauner, D


    Retinal degenerative diseases can have many possible causes and are currently difficult to treat. As an alternative to therapies that require genetic manipulation or the implantation of electronic devices, photopharmacology has emerged as a viable approach to restore visual responses. Here, we present a new photopharmacological strategy that relies on a photoswitchable excitatory amino acid, ATA. This freely diffusible molecule selectively activates AMPA receptors in a light-dependent fashion. It primarily acts on amacrine and retinal ganglion cells, although a minor effect on bipolar cells has been observed. As such, it complements previous pharmacological approaches based on photochromic channel blockers and increases the potential of photopharmacology in vision restoration.

  4. Methylphenidate amplifies long-term potentiation in rat hippocampus CA1 area involving the insertion of AMPA receptors by activation of β-adrenergic and D1/D5 receptors. (United States)

    Rozas, C; Carvallo, C; Contreras, D; Carreño, M; Ugarte, G; Delgado, R; Zeise, M L; Morales, B


    Methylphenidate (MPH, Ritalin©) is widely used in the treatment of Attention Deficit Hyperactivity Disorder and recently as a drug of abuse. Although the effect of MPH has been studied in brain regions such as striatum and prefrontal cortex (PFC), the hippocampus has received relatively little attention. It is known that MPH increases the TBS-dependent Long Term Potentiation (LTP) in the CA1 area. However, the cellular and molecular mechanisms involved in this process are still unknown. Using field potential recordings and western blot analysis in rat hippocampal slices of young rats, we found that acute application of MPH enhances LTP in CA3-CA1 synapses in a dose-dependent manner with an EC50 of 73.44±6.32 nM. Using specific antagonists and paired-pulse facilitation protocols, we observed that the MPH-dependent increase of LTP involves not only β-adrenergic receptors activation but also post-synaptic D1/D5 dopamine receptors. The inhibition of PKA with PKI, suppressed the facilitation of LTP induced by MPH consistent with an involvement of the adenyl cyclase-cAMP-PKA dependent cascade downstream of the activation of D1/D5 receptors. In addition, samples of CA1 areas taken from slices potentiated with MPH presented an increase in the phosphorylation of the Ser845 residue of the GluA1 subunit of AMPA receptors compared to control slices. This effect was reverted by SCH23390, antagonist of D1/D5 receptors, and PKI. Moreover, we found an increase of surface-associated functional AMPA receptors. We propose that MPH increases TBS-dependent LTP in CA3-CA1 synapses through a polysynaptic mechanism involving activation of β-adrenergic and D1/D5 dopaminergic receptors and promoting the trafficking and insertion of functional AMPA receptors to the plasma membrane.

  5. Evidence for loss of synaptic AMPA receptors in anterior piriform cortex of aged mice. (United States)

    Gocel, James; Larson, John


    It has been suggested that age-related impairments in learning and memory may be due to age-related deficits in long-term potentiation of glutamatergic synaptic transmission. For example, olfactory discrimination learning is significantly affected by aging in mice and this may be due, in part, to diminished synaptic plasticity in piriform cortex. In the present study, we tested for alterations in electrophysiological properties and synaptic transmission in this simple cortical network. Whole-cell recordings were made from principal neurons in slices of anterior piriform cortex from young (3-6 months old) and old (24-28 months) C57Bl/6 mice. Miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were collected from cells in presence of tetrodotoxin (TTX) and held at -80 mV in voltage-clamp. Amplitudes of mEPSCs were significantly reduced in aged mice, suggesting that synaptic AMPA receptor expression is decreased during aging. In a second set of experiments, spontaneous excitatory postsynaptic currents (s/mEPSCs) were recorded in slices from different cohorts of young and old mice, in the absence of TTX. These currents resembled mEPSCs and were similarly reduced in amplitude in old mice. The results represent the first electrophysiological evidence for age-related declines in glutamatergic synaptic function in the mammalian olfactory system.

  6. A single high dose of dexamethasone affects the phosphorylation state of glutamate AMPA receptors in the human limbic system (United States)

    Lopes, M W; Leal, R B; Guarnieri, R; Schwarzbold, M L; Hoeller, A; Diaz, A P; Boos, G L; Lin, K; Linhares, M N; Nunes, J C; Quevedo, J; Bortolotto, Z A; Markowitsch, H J; Lightman, S L; Walz, R


    Glucocorticoids (GC) released during stress response exert feedforward effects in the whole brain, but particularly in the limbic circuits that modulates cognition, emotion and behavior. GC are the most commonly prescribed anti-inflammatory and immunosuppressant medication worldwide and pharmacological GC treatment has been paralleled by the high incidence of acute and chronic neuropsychiatric side effects, which reinforces the brain sensitivity for GC. Synapses can be bi-directionally modifiable via potentiation (long-term potentiation, LTP) or depotentiation (long-term depression, LTD) of synaptic transmission efficacy, and the phosphorylation state of Ser831 and Ser845 sites, in the GluA1 subunit of the glutamate AMPA receptors, are a critical event for these synaptic neuroplasticity events. Through a quasi-randomized controlled study, we show that a single high dexamethasone dose significantly reduces in a dose-dependent manner the levels of GluA1-Ser831 phosphorylation in the amygdala resected during surgery for temporal lobe epilepsy. This is the first report demonstrating GC effects on key markers of synaptic neuroplasticity in the human limbic system. The results contribute to understanding how GC affects the human brain under physiologic and pharmacologic conditions. PMID:27959333

  7. Effects of 2,3-benzodiazepine AMPA receptor antagonists on dopamine turnover in the striatum of rats with experimental parkinsonism. (United States)

    Megyeri, Katalin; Marko, Bernadett; Sziray, Nora; Gacsalyi, Istvan; Juranyi, Zsolt; Levay, Gyorgy; Harsing, Laszlo G


    Although levodopa is the current "gold standard" for treatment of Parkinson's disease, there has been disputation on whether AMPA receptor antagonists can be used as adjuvant therapy to improve the effects of levodopa. Systemic administration of levodopa, the precursor of dopamine, increases brain dopamine turnover rate and this elevated turnover is believed to be essential for successful treatment of Parkinson's disease. However, long-term treatment of patients with levodopa often leads to development of dyskinesia. Therefore, drugs that feature potentiation of dopamine turnover rate and are able to reduce daily levodopa dosages might be used as adjuvant in the treatment of patients suffering from Parkinson's disease. To investigate such combined treatment, we have examined the effects of two non-competitive AMPA receptor antagonists, GYKI-52466 and GYKI-53405, alone or in combination with levodopa on dopamine turnover rate in 6-hydroxydopamine-lesioned striatum of the rat. We found here that repeated administration of levodopa, added with the peripheral DOPA decarboxylase inhibitor carbidopa, increased dopamine turnover rate after lesioning the striatum with 6-hydroxydopamine. Moreover, combination of levodopa with GYKI-52466 or GYKI-53405 further increased dopamine turnover enhanced by levodopa administration while the AMPA receptor antagonists by themselves failed to influence striatal dopamine turnover. We concluded from the present data that potentiation observed between levodopa and AMPA receptor antagonists may reflect levodopa-sparing effects in clinical treatment indicating the therapeutic potential of such combination in the management of Parkinson's disease.

  8. Identification of an ionotropic glutamate receptor AMPA1/GRIA1 polymorphism in crossbred beef cows differing in fertility (United States)

    A proposed functional polymorphism in the ionotropic glutamate receptor AMPA1 (GRIA1) has been reported to influence antral follicle numbers and fertility in cows. Repeat Breeder cows that fail to produce a calf in multiple seasons have been reported to have reduced numbers of small (1-3 mm) antral ...

  9. Synthesis and in vitro pharmacology at AMPA and kainate preferring glutamate receptors of 4-heteroarylmethylidene glutamate analogues

    DEFF Research Database (Denmark)

    Valgeirsson, Jon; Christensen, Jeppe K; Kristensen, Anders S;


    2-Amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (1) is a potent AMPA receptor agonist with moderate affinity for native kainic acid (KA) receptors, whereas (S)-E-4-(2,2-dimethylpropylidene)glutamic acid (3) show high affinity for the GluR5 subtype of KA receptors and much lower...... affinity for the GluR2 subtype of AMPA receptors. As an attempt to develop new pharmacological tools for studies of GluR5 receptors, (S)-E-4-(2-thiazolylmethylene)glutamic acid (4a) was designed as a structural hybrid between 1 and 3. 4a was shown to be a potent GluR5 agonist and a high affinity ligand...

  10. PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca(2+)-permeable AMPA receptors. (United States)

    Gerace, E; Masi, A; Resta, F; Felici, R; Landucci, E; Mello, T; Pellegrini-Giampietro, D E; Mannaioni, G; Moroni, F


    An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG, 100μM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD(+) and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca(2+)-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca(2+) permeability of AMPA channels, thus affecting

  11. The AAA+ ATPase, Thorase Regulates AMPA Receptor-Dependent Synaptic Plasticity and Behavior (United States)

    Zhang, Jianmin; Wang, Yue; Chi, Zhikai; Keuss, Matthew J.; Pai, Ying-Min Emily; Kang, Ho Chul; Shin, Jooho; Bugayenko, Artem; Wang, Hong; Xiong, Yulan; Pletnikov, Mikhail V.; Mattson, Mark P.; Dawson, Ted M.; Dawson, Valina L.


    SUMMARY The synaptic insertion or removal of AMPA receptors (AMPAR) plays critical roles in the regulation of synaptic activity reflected in the expression of long-term potentiation (LTP) and long-term depression (LTD). The cellular events underlying this important process in learning and memory are still being revealed. Here we describe and characterize the AAA+ ATPase, Thorase, that regulates the expression of surface AMPAR. In an ATPase-dependent manner Thorase mediates the internalization of AMPAR by disassembling the AMPAR-GRIP1 complex. Following genetic deletion of Thorase, the internalization of AMPAR is substantially reduced, leading to increased amplitudes of miniature excitatory postsynaptic currents, enhancement of LTP and elimination of LTD. These molecular events are expressed as deficits in learning and memory in Thorase null mice. This study identifies an AAA+ ATPase that plays a critical role in regulating the surface expression of AMPAR and thereby regulates synaptic plasticity and learning and memory. PMID:21496646

  12. The effect of AMPA receptor blockade on spatial information acquisition, consolidation and expression in juvenile rats. (United States)

    Tzakis, Nikolaos; Bosnic, Tim; Ritchie, Thomas; Dixon, Kaylyn; Holahan, Matthew R


    Improvement on spatial tasks in rats is observed during a late, postnatal developmental period (post-natal day (PND) 18 - PND 20). The developmental emergence of this spatial function occurs in conjunction with hippocampal connectivity changes and enhanced hippocampal-AMPA receptor-mediated synaptic responses. The current work investigated the effect of AMPAr blockade on the emergence and long-term storage of spatial information in juvenile rats and associated neural activity patterns in the dorsal hippocampus CA1 region. Male, Long Evans rats between the ages of PND 18 and PND 20 were systemically (i.p.) administered the AMPAr antagonist, NBQX, (0, 5 or 10mg/kg) every day prior to hidden platform water maze training (PND 18, 19 and 20), every day immediately post-training or immediately before the probe test (PND 41). NBQX administration prior to training prolonged latencies, pathlength and increased thigmotaxis during the acquisition phase. Administration of NBQX immediately posttraining had no effect on the day-to-day performance. When given a probe test 3weeks later, the saline group across all conditions spent more time in the target quadrant. Rats treated with pretraining 5mg NBQX dose showed a preference for the target quadrant while the posttraining and pretesting 5mg NBQX doses impaired the target quadrant preference. Groups injected with 10mg of NBQX pretraining, posttraining or pretesting did not show a preference for the target quadrant. c-Fos labeling in the CA1 reflected these differences in probe performance in that groups showing greater than chance dwell time in the target quadrant showed more c-Fos labeling in the CA1 region than groups that did not show a target quadrant preference. These findings provide support for the critical role of AMPA receptor-mediated function in the organization and long-term storage of spatial memories acquired during the juvenile period.

  13. [EFfect of quinazolone-alkyl-carboxylic acid derivatives on the transmembrane Ca2+ ion flux mediated by AMPA receptors]. (United States)

    Szárics, Eva; LaszTóczi, Bálint; Nyikos, Lajos; Barabás, Péter; Kovács, Ilona; Skuban, Nina; Nagy, Péter I; Kökösi, József; Takácsné, Novák Krisztina; Kardos, Julianna


    The excitatory neurotransmitter, Glu, plays a crucial role in many sensory and motor functions as well as in brain development, learning and memory and it is also involved in the pathogenesis of a number of neurological disorders, including epilepsy, Alzheimer's and Parkinson's diseases. Therefore, the study of Glu receptors (GluRs) is of therapeutical importance. We showed here by fluorescence monitoring of transmembrane Ca2+ ion fluxes in response to (S)-alpha-amino-3-hidroxi-5-metil-4-izoxazol propionic acid ((S)-AMPA) on the time scale of 0.00004-10 s that Ca2+ ion influx proceeds through faster and slower desensitizing receptors. Pharmacological isolation of the slower and faster desensitizing AMPA receptor was possible by fluorescence monitoring of Ca2+ ion translocation in response to (S)-AMPA in the presence and absence of various 2-methyl-4-oxo-3H-quinazoline-3-alkyl-carboxilic acid derivatives (Qxs): the acetic acid Q1 inhibits the slower desensitizing receptor response specifically, while the acetyl-piperidine Q5 is a more potent inhibitor of the faster desensitizing receptor response. In addition, spontaneous interictal activity, as induced by high [K+] conditions in hippocampal slices, was reduced significantly by Q5, suggesting a possible anticonvulsant property of Q5. Substitutions of Qxs into the GluR2 S1S2 binding core were consistent with their effect by causing variable degree of S1S2 bridging interaction as one of the main determinants of AMPA receptor agonist activity. The exploitation of differences between similar receptors will be important in the development and use of drugs with high pharmacological specificity.

  14. 5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission. (United States)

    Costa, L; Trovato, C; Musumeci, S A; Catania, M V; Ciranna, L


    We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the

  15. Functional characterization of Tet-AMPA [tetrazolyl-2-amino-3-(3-hydroxy-5-methyl- 4-isoxazolyl)propionic acid] analogues at ionotropic glutamate receptors GluR1-GluR4. The molecular basis for the functional selectivity profile of 2-Bn-Tet-AMPA

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Christesen, Thomas; Bølcho, Ulrik;


    Four 2-substituted Tet-AMPA [Tet = tetrazolyl, AMPA = 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid] analogues were characterized functionally at the homomeric AMPA receptors GluR1i, GluR2Qi, GluR3i, and GluR4i in a Fluo-4/Ca2+ assay. Whereas 2-Et-Tet-AMPA, 2-Pr-Tet-AMPA, and 2-i......Pr-Tet-AMPA were nonselective GluR agonists, 2-Bn-Tet-AMPA exhibited a 40-fold higher potency at GluR4i than at GluR1i. Examination of homology models of the S1-S2 domains of GluR1 and GluR4 containing 2-Bn-Tet-AMPA suggested four nonconserved residues in a region adjacent to the orthosteric site as possible...

  16. Blockade of the AMPA receptor prevents CA1 hippocampal injury following severe but transient forebrain ischemia in adult rats. (United States)

    Buchan, A M; Li, H; Cho, S; Pulsinelli, W A


    The cytoprotective effect of NBQX, a selective AMPA receptor antagonist, was tested following 10 min of severe forebrain ischemia using the 4-vessel occlusion model. Immediately, and at 15 and 30 min following reperfusion, adult Wistar rats received intraperitoneal injections of either saline (n = 5), 1 mg lithium chloride (n = 17) or 30 mg/kg of the lithium salt of NBQX (n = 18). In saline-treated animals 82 +/- 12% of CA1 hippocampal neurons were lost. Of those treated with lithium 70 +/- 23% were injured, while those given NBQX sustained only 40 +/- 34% CA1 necrosis (P less than 0.01). Twelve of 18 NBQX-treated animals had less than 30% CA1 injury as compared with 1 of 17 lithium-treated animals. The AMPA receptor may play a more important role than the NMDA receptor in selective ischemic necrosis of hippocampal neurons.

  17. Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors. (United States)

    Dennis, Siobhan H; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G; Henley, Jeremy M; Mellor, Jack R


    Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca(2+), resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A(3) receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca(2+) also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A(3) receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.

  18. AMPA Receptor Phosphorylation and Synaptic Colocalization on Motor Neurons Drive Maladaptive Plasticity below Complete Spinal Cord Injury. (United States)

    Huie, J Russell; Stuck, Ellen D; Lee, Kuan H; Irvine, Karen-Amanda; Beattie, Michael S; Bresnahan, Jacqueline C; Grau, James W; Ferguson, Adam R


    Clinical spinal cord injury (SCI) is accompanied by comorbid peripheral injury in 47% of patients. Human and animal modeling data have shown that painful peripheral injuries undermine long-term recovery of locomotion through unknown mechanisms. Peripheral nociceptive stimuli induce maladaptive synaptic plasticity in dorsal horn sensory systems through AMPA receptor (AMPAR) phosphorylation and trafficking to synapses. Here we test whether ventral horn motor neurons in rats demonstrate similar experience-dependent maladaptive plasticity below a complete SCI in vivo. Quantitative biochemistry demonstrated that intermittent nociceptive stimulation (INS) rapidly and selectively increases AMPAR subunit GluA1 serine 831 phosphorylation and localization to synapses in the injured spinal cord, while reducing synaptic GluA2. These changes predict motor dysfunction in the absence of cell death signaling, suggesting an opportunity for therapeutic reversal. Automated confocal time-course analysis of lumbar ventral horn motor neurons confirmed a time-dependent increase in synaptic GluA1 with concurrent decrease in synaptic GluA2. Optical fractionation of neuronal plasma membranes revealed GluA2 removal from extrasynaptic sites on motor neurons early after INS followed by removal from synapses 2 h later. As GluA2-lacking AMPARs are canonical calcium-permeable AMPARs (CP-AMPARs), their stimulus- and time-dependent insertion provides a therapeutic target for limiting calcium-dependent dynamic maladaptive plasticity after SCI. Confirming this, a selective CP-AMPAR antagonist protected against INS-induced maladaptive spinal plasticity, restoring adaptive motor responses on a sensorimotor spinal training task. These findings highlight the critical involvement of AMPARs in experience-dependent spinal cord plasticity after injury and provide a pharmacologically targetable synaptic mechanism by which early postinjury experience shapes motor plasticity.

  19. Association of the AMPA receptor-related postsynaptic density proteins GRIP and ABP with subsets of glutamate-sensitive neurons in the rat retina. (United States)

    Gábriel, Robert; de Souza, Sunita; Ziff, Edward B; Witkovsky, Paul


    We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain.

  20. Oxygen/glucose Deprivation Induces a Reduction in Synaptic AMPA Receptors on Hippocampal CA3 Neurons Mediated by mGluR1 and A3 Receptors (United States)

    Dennis, Siobhan H.; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G.; Henley, Jeremy M.; Mellor, Jack R.


    Summary Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighbouring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+ resulting in delayed cell death. However, it is unclear if the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 minute OGD protocol a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by mGluR1 or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalisation of AMPARs after OGD. We also show a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists indicating that AMPARs are degraded following internalisation. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection. PMID:21849555

  1. Forster Resonance Energy Transfer (FRET) Analysis of Dual CFP/YFP Labeled AMPA Receptors Reveals Structural Rearrangement within the C-Terminal Domain during Receptor Activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Mila; Plested, Andrew;


    AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. AMPARs are formed by homo- or heterotetramers of GluA1 to GluA4 sub- units. A recent X-ray crystal structure of a full-length homomeric GluA2 A...

  2. Activation of AMPA receptors in the suprachiasmatic nucleus phase-shifts the mouse circadian clock in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Yasutaka Mizoro

    Full Text Available The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor is well elucidated, much less is known about a role of AMPAR (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN.

  3. Modulation of NMDA and AMPA-mediated synaptic transmission by CB1 receptors in frontal cortical pyramidal cells. (United States)

    Li, Qiang; Yan, Haidun; Wilson, Wilkie A; Swartzwelder, H Scott


    Although the endogenous cannabinoid system modulates a variety of physiological and pharmacological processes, the specific role of cannabinoid CB1 receptors in the modulation of glutamatergic neurotransmission and neural plasticity is not well understood. Using whole-cell patch clamp recording techniques, evoked or spontaneous excitatory postsynaptic currents (eEPSCs or sEPSCs) were recorded from visualized, layer II/III pyramidal cells in frontal cortical slices from rat brain. Bath application of the CB1 receptor agonist, WIN 55212-2 (WIN), reduced the amplitude of NMDA receptor-mediated EPSCs in a concentration-dependent manner. When co-applied with the specific CB1 antagonists, AM251 or AM281, WIN did not suppress NMDA receptor-mediated EPSCs. WIN also reduced the amplitude of evoked AMPA receptor-mediated EPSCs, an effect that was also reversed by AM251. Both the frequency and amplitude of spontaneous AMPA receptor-mediated EPSCs were significantly reduced by WIN. In contrast, WIN reduced the frequency, but not the amplitude of miniature EPSCs, suggesting that the suppression of glutamatergic activity by CB1 receptors in the frontal neocortex is mediated by a presynaptic mechanism. Taken together, these data indicate a critical role for endocannabinoid signaling in the regulation of excitatory synaptic transmission in frontal neocortex, and suggest a possible neuronal mechanism whereby THC regulates cortical function.

  4. AMPA receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke. (United States)

    Clarkson, Andrew N; Overman, Justine J; Zhong, Sheng; Mueller, Rudolf; Lynch, Gary; Carmichael, S Thomas


    Stroke is the leading cause of adult disability. Recovery after stroke shares similar molecular and cellular properties with learning and memory. A main component of learning-induced plasticity involves signaling through AMPA receptors (AMPARs). We systematically tested the role of AMPAR function in motor recovery in a mouse model of focal stroke. AMPAR function controls functional recovery beginning 5 d after the stroke. Positive allosteric modulators of AMPARs enhance recovery of limb control when administered after a delay from the stroke. Conversely, AMPAR antagonists impair motor recovery. The contributions of AMPARs to recovery are mediated by release of brain-derived neurotrophic factor (BDNF) in periinfarct cortex, as blocking local BDNF function in periinfarct cortex blocks AMPAR-mediated recovery and prevents the normal pattern of motor recovery. In contrast to a delayed AMPAR role in motor recovery, early administration of AMPAR agonists after stroke increases stroke damage. These findings indicate that the role of glutamate signaling through the AMPAR changes over time in stroke: early potentiation of AMPAR signaling worsens stroke damage, whereas later potentiation of the same signaling system improves functional recovery.

  5. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats. (United States)

    Pellegrini-Giampietro, D E; Zukin, R S; Bennett, M V; Cho, S; Pulsinelli, W A


    Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.

  6. GABA receptor subunit composition relative to insecticide potency and selectivity. (United States)

    Ratra, G S; Casida, J E


    Three observations on the 4-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) binding site in the gamma-aminobutyric acid (GABA) receptor indicate the specific target for insecticide action in human brain and a possible mechanism for selectivity. First, from published data, alpha-endosulfan, lindane and fipronil compete for the [(3)H]EBOB binding site with affinities of 0.3--7 nM in both human recombinant homooligomeric beta 3 receptors and housefly head membranes. Second, from structure-activity studies, including new data, GABAergic insecticide binding potency on the pentameric receptor formed from the beta 3 subunit correlates well with that on the housefly receptor (r=0.88, n=20). This conserved inhibitor specificity is consistent with known sequence homologies in the housefly GABA receptor and the human GABA(A) receptor beta 3 subunit. Third, as mostly new findings, various combinations of alpha 1, alpha 6, and gamma 2 subunits coexpressed with a beta 1 or beta 3 subunit confer differential insecticide binding sensitivity, particularly to fipronil, indicating that subunit composition is a major factor in insecticide selectivity.

  7. Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death. (United States)

    Rameau, Gerald A; Tukey, David S; Garcin-Hosfield, Elsa D; Titcombe, Roseann F; Misra, Charu; Khatri, Latika; Getzoff, Elizabeth D; Ziff, Edward B


    Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca2+ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 microM) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 microM glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca2+-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS.

  8. Excitatory synapses are stronger in the hippocampus of Rett syndrome mice due to altered synaptic trafficking of AMPA-type glutamate receptors. (United States)

    Li, Wei; Xu, Xin; Pozzo-Miller, Lucas


    Deficits in long-term potentiation (LTP) at central excitatory synapses are thought to contribute to cognitive impairments in neurodevelopmental disorders associated with intellectual disability and autism. Using the methyl-CpG-binding protein 2 (Mecp2) knockout (KO) mouse model of Rett syndrome, we show that naïve excitatory synapses onto hippocampal pyramidal neurons of symptomatic mice have all of the hallmarks of potentiated synapses. Stronger Mecp2 KO synapses failed to undergo LTP after either theta-burst afferent stimulation or pairing afferent stimulation with postsynaptic depolarization. On the other hand, basal synaptic strength and LTP were not affected in slices from younger presymptomatic Mecp2 KO mice. Furthermore, spine synapses in pyramidal neurons from symptomatic Mecp2 KO are larger and do not grow in size or incorporate GluA1 subunits after electrical or chemical LTP. Our data suggest that LTP is occluded in Mecp2 KO mice by already potentiated synapses. The higher surface levels of GluA1-containing receptors are consistent with altered expression levels of proteins involved in AMPA receptor trafficking, suggesting previously unidentified targets for therapeutic intervention for Rett syndrome and other MECP2-related disorders.

  9. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells. (United States)

    Liu, Yu; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June


    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca(2+) and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the duration of action potentials can suppress the expression of GluR2-containing AMPARs at stellate cell synapses. We find that large-conductance Ca(2+)-activated potassium (BK) channels mediate a large proportion of the depolarization-evoked noninactivating potassium current in stellate cells. Pharmacological blockade of BK channels prolonged the action potential duration in postsynaptic stellate cells and altered synaptic AMPAR subtype from GluR2-lacking to GluR2-containing Ca(2+)-impermeable AMPARs. An L-type channel blocker abolished an increase in Ca(2+) entry that was associated with spike broadening and also prevented the BK channel blocker-induced switch in AMPAR phenotype. Thus blocking BK potassium channels prolongs the action potential duration and increases the expression of GluR2-containing receptors at the synapse by enhancing Ca(2+) entry in cerebellar stellate cells.

  10. AMPA receptor trafficking in inflammation-induced dorsal horn central sensitization

    Institute of Scientific and Technical Information of China (English)

    Yuan-Xiang Tao


    Activity-dependent postsynaptic receptor trafficking is critical for long-term synaptic plasticity in the brain,but it is unclear whether this mechanism actually mediates the spinal cord dorsal horn central sensitization (a specific form of synaptic plasticity) that is associated with persistent pain.Recent studies have shown that peripheral inflammation drives changes in α-amino-3-hydroxy-5-methy1-4-isoxazolepropionic acid receptor (AMPAR) subunit trafficking in the dorsal horn and that such changes contribute to the hypersensitivity that underlies persistent pain.Here,we review current evidence to illustrate how spinal cord AMPARs participate in the dorsal horn central sensitization associated with persistent pain.Understanding these mechanisms may allow the development of novel therapeutic strategies for treating persistent pain.

  11. Domain architecture of a calcium-permeable AMPA receptor in a ligand-free conformation

    Directory of Open Access Journals (Sweden)

    Charles R. Midgett


    Full Text Available Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs, the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD, and of multiple conformations of the ligand-binding domain (LBD. Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPARs, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation.

  12. A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures

    DEFF Research Database (Denmark)

    Bundgaard Christiansen, Gitte; Harbak, Barbara; E. Hede, Susanne;


    -mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two...... information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing...

  13. Phenobarbital but not diazepam reduces AMPA/Kainate receptor mediated currents and exerts opposite actions on initial seizures in the neonatal rat hippocampus

    Directory of Open Access Journals (Sweden)

    Romain eNardou


    Full Text Available Diazepam (DZP and phenobarbital (PB are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. Yet, their efficacy is variable with occasional failure or even aggravation of recurrent seizures questioning whether other mechanisms are not involved in their actions. We have now compared the effects of DZP and PB on ictal-like events (ILEs in an in vitro model of mirror focus (MF. Using the three-compartment chamber with the two immature hippocampi and their commissural fibers placed in 3 different compartments, kainate was applied to one hippocampus and PB or DZP to the contralateral one, either after one ILE or after many recurrent ILEs that produce an epileptogenic MF. We report that in contrast to PB, DZP aggravated propagating ILEs from the start and did not prevent the formation of MF. PB reduced and DZP increased the network driven Giant Depolarising Potentials suggesting that PB may exert additional actions that are not mediated by GABA signalling. In keeping with this, PB but not DZP reduced field potentials recorded in the presence of GABA and NMDA receptor antagonists. These effects are mediated by a direct action on AMPA/Kainate receptors since PB: i reduced AMPA/Kainate receptor mediated currents induced by focal applications of glutamate ; ii reduced the amplitude and the frequency of AMPA but not NMDA receptor mediated miniature EPSCs; iii augmented the number of AMPA receptor mediated EPSCs failures evoked by minimal stimulation. These effects persisted in MF. Therefore, PB exerts its anticonvulsive actions partly by reducing AMPA/Kainate receptors mediated EPSCs in addition to the pro-GABA effects. We suggest that PB may have advantage over DZP in the treatment of initial neonatal seizures since the additional reduction of glutamate receptors mediated signals may reduce the severity of neonatal seizures.

  14. Role of AMPA and GluR5 kainate receptors in the development and expression of amygdala kindling in the mouse. (United States)

    Rogawski, M A; Kurzman, P S; Yamaguchi, S I; Li, H


    The role of AMPA and GluR5-containing kainate receptors in the development and expression of amygdala kindling was examined using the selective 2,3-benzodiazepine AMPA receptor antagonist GYKI 52466 [(1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine] and the decahydroisoquinoline mixed AMPA receptor and GluR5 kainate receptor antagonist LY293558 {(3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline- 3-carboxy lic acid)}. Administration of GYKI 52466 (5-40 mg/kg, intraperitoneally) and LY293558 (10-40 mg/kg, intraperitoneally) prior to daily kindling stimulation in mice produced a dose-dependent suppression of the rate of development of behavioral kindled seizure activity and reduced the duration of the stimulation-induced electrographic afterdischarge. In drug-free stimulation sessions after the initial drug-treatment sessions, there was an acceleration in the rate of kindling development compared with the rate during the preceding drug-administration period; the "rebound" rate was also greater than the kindling rate in saline-treated control animals. In fully kindled animals, both GYKI 52466 and LY293558 produced a dose-dependent suppression of evoked seizures (ED(50), 19.3 and 16.7 mg/kg, respectively). Although AMPA receptors appear to be critical to the expression of kindled seizures, since kindling development progressed despite the suppression of behavioral seizure activity, AMPA receptors are less important to the kindling process. LY293558 was modestly less effective at suppressing behavioral seizures during kindling and was not superior to GYKI 52466 in retarding the overall extent of kindling development, indicating that GluR5 kainate receptors do not contribute to epileptogenesis in this model.

  15. Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures. (United States)

    Kristensen, B W; Noraberg, J; Zimmer, J


    The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration

  16. A new phenylalanine derivative acts as an antagonist at the AMPA receptor GluA2 and introduces partial domain closure

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Frydenvang, Karla; Contreras-Sanz, Alberto;


    In order to map out molecular determinants for competitive blockade of AMPA receptor subtypes, a series of 2-carboxyethylphenylalanine derivatives has been synthesized and pharmacologically characterized in vitro. One compound in this series, (RS)-3h, showed micromolar affinity for GluA1(o) and G...

  17. Studies on an (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonist IKM-159

    DEFF Research Database (Denmark)

    Juknaite, Lina; Sugamata, Yutaro; Tokiwa, Kazuya;


    IKM-159 was developed and identified as a member of a new class of heterotricyclic glutamate analogs that act as AMPA receptor-selective antagonists. However, it was not known which enantiomer of IKM-159 was responsible for its pharmacological activities. Here, we report in vivo and in vitro neur...

  18. Enhanced odor discrimination and impaired olfactory memory by spatially controlled switch of AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Derya R Shimshek


    Full Text Available Genetic perturbations of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs are widely used to dissect molecular mechanisms of sensory coding, learning, and memory. In this study, we investigated the role of Ca2+-permeable AMPARs in olfactory behavior. AMPAR modification was obtained by depletion of the GluR-B subunit or expression of unedited GluR-B(Q, both leading to increased Ca2+ permeability of AMPARs. Mice with this functional AMPAR switch, specifically in forebrain, showed enhanced olfactory discrimination and more rapid learning in a go/no-go operant conditioning task. Olfactory memory, however, was dramatically impaired. GluR-B depletion in forebrain was ectopically variable ("mosaic" among individuals and strongly correlated with decreased olfactory memory in hippocampus and cortex. Accordingly, memory was rescued by transgenic GluR-B expression restricted to piriform cortex and hippocampus, while enhanced odor discrimination was independent of both GluR-B variability and transgenic GluR-B expression. Thus, correlated differences in behavior and levels of GluR-B expression allowed a mechanistic and spatial dissection of olfactory learning, discrimination, and memory capabilities.

  19. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)


    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  20. Anti-AMPA-Receptor Encephalitis Presenting as a Rapid-Cycling Bipolar Disorder in a Young Woman with Turner Syndrome

    Directory of Open Access Journals (Sweden)

    Giuseppe Quaranta


    Full Text Available Background. Autoimmune encephalitis is a disorder characterised by the subacute onset of seizures, short-term memory loss, and psychiatric and behavioural symptoms. Initially, it was recognised as a paraneoplastic disorder, but recently a subgroup of patients without systemic cancer was identified. Case Description. We describe a 20-year-old woman with Turner syndrome presenting with a treatment-resistant rapid cycling bipolar disorder with cognitive impairment. She was diagnosed with anti-AMPA-receptor encephalitis. She showed marked improvement after starting memantine and valproic acid. Conclusion. This case description emphasises the importance of timely recognition of autoimmune limbic encephalitis in patients with psychiatric manifestations and a possible predisposition to autoimmune conditions, in order to rule out malignancy and to quickly initiate treatment.

  1. Persistent inflammation-induced up-regulation of brain-derived neurotrophic factor (BDNF) promotes synaptic delivery of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA1 subunits in descending pain modulatory circuits. (United States)

    Tao, Wenjuan; Chen, Quan; Zhou, Wenjie; Wang, Yunping; Wang, Lu; Zhang, Zhi


    The enhanced AMPA receptor phosphorylation at GluA1 serine 831 sites in the central pain-modulating system plays a pivotal role in descending pain facilitation after inflammation, but the underlying mechanisms remain unclear. We show here that, in the rat brain stem, in the nucleus raphe magnus, which is a critical relay in the descending pain-modulating system of the brain, persistent inflammatory pain induced by complete Freund adjuvant (CFA) can enhance AMPA receptor-mediated excitatory postsynaptic currents and the GluA2-lacking AMPA receptor-mediated rectification index. Western blot analysis showed an increase in GluA1 phosphorylation at Ser-831 but not at Ser-845. This was accompanied by an increase in distribution of the synaptic GluA1 subunit. In parallel, the level of histone H3 acetylation at bdnf gene promoter regions was reduced significantly 3 days after CFA injection, as indicated by ChIP assays. This was correlated with an increase in BDNF mRNA levels and BDNF protein levels. Sequestering endogenous extracellular BDNF with TrkB-IgG in the nucleus raphe magnus decreased AMPA receptor-mediated synaptic transmission and GluA1 phosphorylation at Ser-831 3 days after CFA injection. Under the same conditions, blockade of TrkB receptor functions, phospholipase C, or PKC impaired GluA1 phosphorylation at Ser-831 and decreased excitatory postsynaptic currents mediated by GluA2-lacking AMPA receptors. Taken together, these results suggest that epigenetic up-regulation of BDNF by peripheral inflammation induces GluR1 phosphorylation at Ser-831 sites through activation of the phospholipase C-PKC signaling cascade, leading to the trafficking of GluA1 to pain-modulating neuronal synapses.

  2. Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process. (United States)

    Pierre, Karin; Chatton, Jean-Yves; Parent, Annabelle; Repond, Cendrine; Gardoni, Fabrizio; Di Luca, Monica; Pellerin, Luc


    MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

  3. Differential localization of delta glutamate receptors in the rat cerebellum: coexpression with AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses. (United States)

    Landsend, A S; Amiry-Moghaddam, M; Matsubara, A; Bergersen, L; Usami, S; Wenthold, R J; Ottersen, O P


    The delta 2 glutamate receptors are prominently expressed in Purkinje cells and are thought to play a key role in the induction of cerebellar long-term depression. The synaptic and subsynaptic localization of delta receptors in rat cerebellar cortex was investigated with sensitive and high-resolution immunogold procedures. After postembedding incubation with an antibody raised to a C-terminal peptide of delta 2, high gold particle densities occurred in all parallel fiber synapses with Purkinje cell dendritic spines, whereas other synapses were consistently devoid of labeling. Among the types of immunonegative synapse were climbing fiber synapses with spines and parallel fiber synapses with dendritic stems of interneurons. At the parallel fiber-spine synapse, gold particles signaling delta receptors were restricted to the postsynaptic specialization. By the use of double labeling with two different gold particle sizes, it was shown that delta and AMPA GluR2/3 receptors were colocalized along the entire extent of the postsynaptic specialization without forming separate domains. The distribution of gold particles representing delta receptors was consistent with a cytoplasmic localization of the C terminus and an absence of a significant presynaptic pool of receptor molecules. The present data suggest that the delta 2 receptors are targeted selectively to a subset of Purkinje cell spines and that they are coexpressed with ionotropic receptors in the postsynaptic specialization. This arrangement could allow for a direct interaction between the two classes of receptor.

  4. The Prefrontal Dectin-1/AMPA Receptor Signaling Pathway Mediates The Robust and Prolonged Antidepressant Effect of Proteo-β-Glucan from Maitake (United States)

    Bao, Hongkun; Ran, Pengzhan; Zhu, Ming; Sun, Lijuan; Li, Bai; Hou, Yangyang; Nie, Jun; Shan, Liping; Li, Hongliang; Zheng, Shangyong; Xu, Xiufeng; Xiao, Chunjie; Du, Jing


    Proteo-β-glucan from Maitake (PGM) is a strong immune regulator, and its receptor is called Dectin-1. Cumulative evidence suggests that AMPA receptors are important for the treatment of depression. Here, we report that PGM treatment leads to a significant antidepressant effect in the tail suspension test and forced swim test after sixty minutes of treatment in mice. After five consecutive days of PGM treatment, this antidepressant effect remained. PGM treatment did not show a hyperactive effect in the open field test. PGM significantly enhanced the expression of its receptor Dectin-1, as well as p-GluA1(S845) and GluA1, but not GluA2 or GluA3 in the prefrontal cortex (PFC) after five days of treatment. The Dectin-1 inhibitor Laminarin was able to block the antidepressant effect of PGM. At the synapses of PFC, PGM treatment significantly up-regulated the p-GluA1(S845), GluA1, GluA2, and GluA3 levels. Moreover, PGM’s antidepressant effects and the increase of p-GluA1(S845)/GluA1 lasted for 3 days after stopping treatment. The AMPA-specific antagonist GYKI 52466 was able to block the antidepressant effect of PGM. This study identified PGM as a novel antidepressant with clinical potential and a new antidepressant mechanism for regulating prefrontal Dectin-1/AMPA receptor signalling. PMID:27329257

  5. Role of Hippocampal 5-HT1A Receptor and Its Modulation to NMDA Receptor and AMPA Receptor in Depression Induced by Chronic Unpredictable Mild Stress%应激性抑郁样行为发生中海马5-羟色胺1A受体的作用及其对NMDA受体和AMPA受体的调节

    Institute of Scientific and Technical Information of China (English)

    问黎敏; 安书成; 刘慧


    sufficient validity. The most accepted one is the chronic unpredictable mild stress (CUMS) rodent model, in which rats are chronically and unpredictably subjected to a variety of stressors including immersion in cold water, tail pinch, day and night reversal, and so on. There are several theoretical mechanisms for depression, such as the monoamine neurotransmitter imbalance theory and the neural plasticity theory, but none of them can fully elucidate the formation of depression. Due to the weak and irregular anti-depressant effects of monoamines, glutamate (Glu) and its receptors, especially N-methyl-D-aspartic acid (NMDA) receptors and a-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA) receptors, have gained more attention in recent years. As an important isoform of the 5-hydroxytryptamine (5-HT) receptor, the 1A receptor is highly expressed in the hippocampus. Numerous pharmacological and clinical studies show that the 1A receptor is correlated with the development and therapy of major depressive disorder, anxiety, and drug addiction. The present study investigates the expression and role of the 5-HT receptor 1A (5-HT1AR) and its relationship with NMDA and AMPA receptors in depression induced by CUMS.The CUMS induced depression model was done using Sprague-Dawley rats that were given intra-hippocampal microinjections of drugs. The location of injections was determined by rat brain stereotaxic coordinates. The behavioral observations were conducted by measurement of weight changes, sucrose preference test, open-field test, and tail suspension test. The expression of 5-HT1AR was detected by Western blot, and the expression and phosphorylation of the NMDA and AMPA receptor's subunits were detected by Western blot and ELISA, respectively.The results showed that CUMS rats had depressive-like behavior, lower expression of 5-HT1AR, lower expression and phosphorylation of AMPA receptor subunits (GluR2/3), and higher expression and phosphorylation of NMDA receptor

  6. Consolidation of remote fear memories involves Corticotropin-Releasing Hormone (CRH) receptor type 1-mediated enhancement of AMPA receptor GluR1 signaling in the dentate gyrus. (United States)

    Thoeringer, Christoph K; Henes, Kathrin; Eder, Matthias; Dahlhoff, Maik; Wurst, Wolfgang; Holsboer, Florian; Deussing, Jan M; Moosmang, Sven; Wotjak, Carsten T


    Persistent dreadful memories and hyperarousal constitute prominent psychopathological features of posttraumatic stress disorder (PTSD). Here, we used a contextual fear conditioning paradigm to demonstrate that conditional genetic deletion of corticotropin-releasing hormone (CRH) receptor 1 within the limbic forebrain in mice significantly reduced remote, but not recent, associative and non-associative fear memories. Per os treatment with the selective CRHR1 antagonist DMP696 (3 mg/kg) attenuated consolidation of remote fear memories, without affecting their expression and retention. This could be achieved, if DMP696 was administered for 1 week starting as late as 24 h after foot shock. Furthermore, by combining electrophysiological recordings and western blot analyses, we demonstrate a delayed-onset and long-lasting increase in AMPA receptor (AMPAR) GluR1-mediated signaling in the dentate gyrus (DG) of the dorsal hippocampus 1 month after foot shock. These changes were absent from CRHR1-deficient mice and after DMP696 treatment. Inactivation of hippocampal GluR1-containing AMPARs by antisense oligonucleotides or philantotoxin 433 confirmed the behavioral relevance of AMPA-type glutamatergic neurotransmission in maintaining the high levels of remote fear in shocked mice with intact CRHR1 signaling. We conclude that limbic CRHR1 receptors enhance the consolidation of remote fear memories in the first week after foot shock by increasing the expression of Ca(2+)-permeable GluR1-containing AMPARs in the DG. These findings suggest both receptors as rational targets for the prevention and therapy, respectively, of psychopathology associated with exaggerated fear memories, such as PTSD.

  7. RNA editing of the GABAA receptor α3 subunit alters the functional properties of recombinant receptors


    Nimmich, Mitchell L.; Heidelberg, Laura S.; Fisher, Janet L.


    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors co...

  8. Regulated RalBP1 binding to RalA and PSD-95 controls AMPA receptor endocytosis and LTD.

    Directory of Open Access Journals (Sweden)

    Kihoon Han


    Full Text Available Long-term depression (LTD is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR-dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.

  9. Alteration of AMPA Receptor-Mediated Synaptic Transmission by Alexa Fluor 488 and 594 in Cerebellar Stellate Cells. (United States)

    Maroteaux, Matthieu; Liu, Siqiong June


    The fluorescent dyes, Alexa Fluor 488 and 594 are commonly used to visualize dendritic structures and the localization of synapses, both of which are critical for the spatial and temporal integration of synaptic inputs. However, the effect of the dyes on synaptic transmission is not known. Here we investigated whether Alexa Fluor dyes alter the properties of synaptic currents mediated by two subtypes of AMPA receptors (AMPARs) at cerebellar stellate cell synapses. In naive mice, GluA2-lacking AMPAR-mediated synaptic currents displayed an inwardly rectifying current-voltage (I-V) relationship due to blockade by cytoplasmic spermine at depolarized potentials. We found that the inclusion of 100 µm Alexa Fluor dye, but not 10 µm, in the pipette solution led to a gradual increase in the amplitude of EPSCs at +40 mV and a change in the I-V relationship from inwardly rectifying to more linear. In mice exposed to an acute stress, AMPARs switched to GluA2-containing receptors, and 100 µm Alexa Fluor 594 did not alter the I-V relationship of synaptic currents. Therefore, a high concentration of Alexa Fluor dye changed the I-V relationship of EPSCs at GluA2-lacking AMPAR synapses.

  10. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors. (United States)

    Takeuchi, Yusuke; Morise, Jyoji; Morita, Ippei; Takematsu, Hiromu; Oka, Shogo


    The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  11. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    Directory of Open Access Journals (Sweden)

    Yusuke Takeuchi

    Full Text Available The AMPA-type glutamate receptor (AMPAR, which is a tetrameric complex composed of four subunits (GluA1-4 with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc, human natural killer-1 (HNK-1 carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413 within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  12. Activation of AMPA receptor promotes TNF-α release via the ROS-cSrc-NFκB signaling cascade in RAW264.7 macrophages

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    Cheng, Xiu-Li [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Ding, Fan [Office of Scientific R& D, Tsinghua University, Beijing (China); Li, Hui; Tan, Xiao-Qiu [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Liu, Xiao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Cao, Ji-Min, E-mail: [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Gao, Xue, E-mail: [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China)


    The relationship between glutamate signaling and inflammation has not been well defined. This study aimed to investigate the role of AMPA receptor (AMPAR) in the expression and release of tumor necrosis factor-alpha (TNF-α) from macrophages and the underlying mechanisms. A series of approaches, including confocal microscopy, immunofluorescency, flow cytometry, ELISA and Western blotting, were used to estimate the expression of AMPAR and downstream signaling molecules, TNF-α release and reactive oxygen species (ROS) generation in the macrophage-like RAW264.7 cells. The results demonstrated that AMPAR was expressed in RAW264.7 cells. AMPA significantly enhanced TNF-α release from RAW264.7 cells, and this effect was abolished by CNQX (AMPAR antagonist). AMPA also induced elevation of ROS production, phosphorylation of c-Src and activation of nuclear factor (NF)-κB in RAW264.7 cells. Blocking c-Src by PP2, scavenging ROS by glutathione (GSH) or inhibiting NF-κB activation by pyrrolidine dithiocarbamate (PDTC) decreased TNF-α production from RAW264.7 cells. We concluded that AMPA promotes TNF-α release in RAW264.7 macrophages likely through the following signaling cascade: AMPAR activation → ROS generation → c-Src phosphorylation → NF-κB activation → TNF-α elevation. The study suggests that AMPAR may participate in macrophage activation and inflammation. - Highlights: • AMPAR is expressed in RAW264.7 macrophages and is upregulated by AMPA stimulation. • Activation of AMPAR stimulates TNF-α release in macrophages through the ROS-cSrc-NFκB signaling cascade. • Macrophage AMPAR signaling may play an important role in inflammation.

  13. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila


    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  14. Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes. (United States)

    McNair, L F; Kohlmeier, K A


    Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the

  15. Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans. (United States)

    Wu, Ye; Arai, Amy C; Rumbaugh, Gavin; Srivastava, Anand K; Turner, Gillian; Hayashi, Takashi; Suzuki, Erika; Jiang, Yuwu; Zhang, Lilei; Rodriguez, Jayson; Boyle, Jackie; Tarpey, Patrick; Raymond, F Lucy; Nevelsteen, Joke; Froyen, Guy; Stratton, Mike; Futreal, Andy; Gecz, Jozef; Stevenson, Roger; Schwartz, Charles E; Valle, David; Huganir, Richard L; Wang, Tao


    Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans.

  16. Embryonic expression of zebrafish AMPA receptor genes: zygotic gria2alpha expression initiates at the midblastula transition. (United States)

    Lin, Wei-Hsiang; Wu, Chan-Hwa; Chen, Yu-Chia; Chow, Wei-Yuan


    The AMPA-preferring receptors (AMPARs) mediate rapid excitatory synaptic transmission in the central nervous system of vertebrates. Expression profiles of 8 AMPAR genes were studied by RT-PCR analyses to elucidate the properties of AMPARs during early zebrafish development. Transcripts of all AMPAR genes are detected at the time of fertilization, suggesting maternal transcriptions of zebrafish AMPAR genes. The amounts of gria1 and gria2 transcripts are several-fold higher than that of gria3 and gria4 between 10 and 72 hpf (hour postfertilization). The edited gria2alpha transcript decreases during gastrulation period, suggesting that zygotic expression of gria2alpha begins around the time of midblastula transition. Relative to the amount of beta-actin, the amounts of AMPAR transcripts increase significantly after the completion of neurulation. The amounts of gria2 transcripts exceed the total amounts of the remaining AMPAR transcripts after 36 hpf, suggesting increases in the representation of low Ca2+ permeable AMPARs during neuronal maturation. Many but not all of the known mammalian protein-protein interaction motifs are preserved in the C-terminal domains (CTD) of zebrafish AMPARs. Before 16 hpf, the embryos express predominantly the alternative splice forms encoding longer CTD. Representations of the short CTD splice forms of gria2 and gria4alpha increase after 24 hpf, when neurulation is nearly completed.

  17. Activation of AMPA receptor in the infralimbic cortex facilitates extinction and attenuates the heroin-seeking behavior in rats. (United States)

    Chen, Weisheng; Wang, Yiqi; Sun, Anna; Zhou, Linyi; Xu, Wenjin; Zhu, Huaqiang; Zhuang, Dingding; Lai, Miaojun; Zhang, Fuqiang; Zhou, Wenhua; Liu, Huifen


    Infralimbic cortex (IL) is proposed to suppress cocaine seeking after extinction, but whether the IL regulates the extinction and reinstatement of heroin-seeking behavior is unknown. To address this issue, the male SD rats were trained to self-administer heroin under a FR1 schedule for consecutive 14 days, then the rats underwent 7 daily 2h extinction session in the operant chamber. The activation of IL by microinjection PEPA, an allosteric AMPA receptor potentiator into IL before each of extinction session facilitated the extinction responding after heroin self-administration, but did not alter the locomotor activity in an open field testing environment. Other rats were first trained under a FR1 schedule for heroin self-administration for 14 days, followed by 14 days of extinction training, and reinstatement of heroin-seeking induced by cues was measured for 2h. Intra-IL microinjecting of PEPA at 15min prior to test inhibited the reinstatement of heroin-seeking induced by cues. Moreover, the expression of GluR1 in the IL and NAc remarkably increased after treatment with PEPA during the reinstatement. These finding suggested that activation of glutamatergic projection from IL to NAc shell may be involved in the extinction and reinstatement of heroin-seeking.

  18. Synthetic and endogenous cannabinoids protect retinal neurons from AMPA excitotoxicity in vivo, via activation of CB1 receptors: Involvement of PI3K/Akt and MEK/ERK signaling pathways. (United States)

    Kokona, Despina; Thermos, Kyriaki


    Cannabinoids have been suggested to protect retinal ganglion cells in different models of toxicity, but their effects on other retinal neurons are poorly known. We investigated the neuroprotective actions of the endocannabinoid N-arachidonoyl ethanolamine (Anandamide/AEA) and the synthetic cannabinoids R1-Methanandamide (MethAEA) and HU-210, in an in vivo retinal model of AMPA excitotoxicity, and the mechanisms involved in the neuroprotection. Sprague-Dawley rats were intravitreally injected with PBS or AMPA in the absence or presence of the cannabinoid agonists. Brain nitric oxide synthase (bNOS) and choline acetyltransferase (ChAT) immunoreactivity (IR), as well as TUNEL staining, assessed the AMPA-induced retinal amacrine cell loss and the dose-dependent neuroprotection afforded by cannabinoids. The CB1 receptor selective antagonist AM251 and the PI3K/Akt inhibitor wortmannin reversed the cannabinoid-induced neuroprotection, suggesting the involvement of CB1 receptors and the PI3K/Akt pathway in cannabinoids' actions. Experiments with the CB2 agonist JWH015 and [(3)H]CP55940 radioligand binding suggested that the CB2 receptor is not involved in the neuroprotection. AEA and HU-210 induced phosphorylation of Akt but only AEA induced phosphorylation of ERK1/2 kinases, as revealed by western blot analysis. To investigate the role of caspase-3 in the AMPA-induced cell death, the caspase-3 inhibitor Z-DEVD-FMK was co-injected with AMPA. Z-DEVD-FMK had no effect on AMPA excitotoxicity. Moreover, no difference was observed in the phosphorylation of SAPK/JNK kinases between PBS- and AMPA-treated retinas. These results suggest that endogenous and synthetic cannabinoids protect retinal amacrine neurons from AMPA excitotoxicity in vivo via a mechanism involving the CB1 receptors, and the PI3K/Akt and/or MEK/ERK1/2 signaling pathways.

  19. Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus. (United States)

    Didelon, F; Mladinic', M; Cherubini, E; Bradbury, A


    The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

  20. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites

    DEFF Research Database (Denmark)

    Kaae, Birgitte Høiriis; Harpsøe, Kasper; Kastrup, Jette Sandholm Jensen;


    Dimeric positive allosteric modulators of ionotropic glutamate receptors were designed, synthesized, and characterized pharmacologically in electrophysiological experiments. The designed compounds are dimers of arylpropylsulfonamides and have been constructed without a linker. The monomeric...

  1. NMDA and AMPA/kainate glutamatergic receptors in the prelimbic medial prefrontal cortex modulate the elaborated defensive behavior and innate fear-induced antinociception elicited by GABAA receptor blockade in the medial hypothalamus. (United States)

    de Freitas, Renato Leonardo; Salgado-Rohner, Carlos José; Biagioni, Audrey Francisco; Medeiros, Priscila; Hallak, Jaime Eduardo Cecílio; Crippa, José Alexandre S; Coimbra, Norberto Cysne


    The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) and amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptors of the prelimbic (PL) division of the medial prefrontal cortex (MPFC) on the panic attack-like reactions evoked by γ-aminobutyric acid-A receptor blockade in the medial hypothalamus (MH). Rats were pretreated with NaCl 0.9%, LY235959 (NMDA receptor antagonist), and NBQX (AMPA/kainate receptor antagonist) in the PL at 3 different concentrations. Ten minutes later, the MH was treated with bicuculline, and the defensive responses were recorded for 10 min. The antagonism of NMDA receptors in the PL decreased the frequency and duration of all defensive behaviors evoked by the stimulation of the MH and reduced the innate fear-induced antinociception. However, the pretreatment of the PL cortex with NBQX was able to decrease only part of defensive responses and innate fear-induced antinociception. The present findings suggest that the NMDA-glutamatergic system of the PL is critically involved in panic-like responses and innate fear-induced antinociception and those AMPA/kainate receptors are also recruited during the elaboration of fear-induced antinociception and in panic attack-related response. The activation of the glutamatergic neurotransmission of PL division of the MPFC during the elaboration of oriented behavioral reactions elicited by the chemical stimulation of the MH recruits mainly NMDA receptors in comparison with AMPA/kainate receptors.

  2. Regulation of AMPA receptor function by the human memory-associated gene KIBRA. (United States)

    Makuch, Lauren; Volk, Lenora; Anggono, Victor; Johnson, Richard C; Yu, Yilin; Duning, Kerstin; Kremerskothen, Joachim; Xia, Jun; Takamiya, Kogo; Huganir, Richard L


    KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity.

  3. Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor

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    Christoph Straub


    Full Text Available Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

  4. Involvement of AMPA/kainate and GABAA receptors in topiramate neuroprotective effects against methylphenidate abuse sequels involving oxidative stress and inflammation in rat isolated hippocampus. (United States)

    Motaghinejad, Majid; Motevalian, Manijeh


    Abuses of methylphenidate (MPH) as psychostimulant cause neural damage of brain cells. Neuroprotective properties of topiramate (TPM) have been indicated in several studies but its exact mechanism of action remains unclear. The current study evaluates protective role of various doses of TPM and its mechanism of action in MPH induced oxidative stress and inflammation. The neuroprotective effects of various doses of TPM against MPH induced oxidative stress and inflammation were evaluated and then the action of TPM was studied in presence of domoic acid (DOM), as AMPA/kainate receptor agonist and bicuculline (BIC) as GABAA receptor antagonist, in isolated rat hippocampus. Open Field Test (OFT) was used to investigate motor activity changes. Oxidative, antioxidant and inflammatory factors were measured in isolated hippocampus. TPM (70 and 100mg/kg) decreased MPH induced motor activity disturbances and inhibit MPH induced oxidative stress and inflammation. On the other hand pretreatment of animals with DOM or BIC, inhibit this effect of TPM and potentiate MPH induced motor activity disturbances and increased lipid peroxidation, mitochondrial oxidized form of glutathione (GSSG) level, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in isolated hippocampal cells and decreased reduced form of glutathione (GSH) level, superoxide dismutase, glutathione peroxidase and glutathione reductase activity. It seems that TPM can protect cells of hippocampus from oxidative stress and neuroinflammation and it could be partly by activation of GABAA receptor and inhibition of AMPA/kainite receptor.

  5. Central nitric oxide modulates hindquarter vasodilation elicited by AMPA receptor stimulation in the NTS of conscious rats. (United States)

    Dias, Ana Carolina Rodrigues; Colombari, Eduardo


    Microinjection of S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the nucleus of the solitary tract (NTS) of conscious rats causes hypertension, bradycardia, and vasoconstriction in the renal, mesenteric, and hindquarter vascular beds. In the hindquarter, the initial vasoconstriction is followed by vasodilation with AMPA doses >5 pmol/100 nl. To test the hypothesis that this vasodilation is caused by activation of a nitroxidergic pathway in the NTS, we examined the effect of pretreatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10 nmol/100 nl, microinjected into the NTS) on changes in mean arterial pressure, heart rate, and regional vascular conductance (VC) induced by microinjection of AMPA (10 pmol/100 nl in the NTS) in conscious rats. AMPA increased hindquarter VC by 18 +/- 4%, but after pretreatment with L-NAME, AMPA reduced hindquarter VC by 16 +/- 7% and 17 +/- 9% (5 and 15 min after pretreatment, P NTS activates both vasodilatatory and vasoconstrictor mechanisms and that the vasodilatatory mechanism depends on production of nitric oxide in the NTS.

  6. Inhibition of AMPA Receptors by Polyamine Toxins is Regulated by Agonist Efficacy and Stargazin

    DEFF Research Database (Denmark)

    Poulsen, Mette H; Lucas, Simon; Strømgaard, Kristian;


    The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels mediating the majority of fast excitatory synaptic transmission in the central nervous system (CNS). Polyamine toxins derived from spiders and wasps are use- and voltage-dependent...... explored the effect of the TARP γ-2 (also known as stargazin) on the inhibitory potency of three structurally different polyamine toxins at Ca(2+)-permeable homomeric GluA1 AMPARs expressed in oocytes. We find that polyamine toxin IC50 is differentially affected by presence of stargazin depending...... on the efficacy of the agonists used to activate GluA1. Co-assembly of GluA1 receptors with stargazin increases the potency of the polyamine toxins when activated by the weak partial agonist kainate, but has no effect in presence of full-agonist L-glutamate (Glu) and partial agonist (RS)-willardiine....

  7. Acute stress causes rapid synaptic insertion of Ca2+ -permeable AMPA receptors to facilitate long-term potentiation in the hippocampus. (United States)

    Whitehead, Garry; Jo, Jihoon; Hogg, Ellen L; Piers, Thomas; Kim, Dong-Hyun; Seaton, Gillian; Seok, Heon; Bru-Mercier, Gilles; Son, Gi Hoon; Regan, Philip; Hildebrandt, Lars; Waite, Eleanor; Kim, Byeong-Chae; Kerrigan, Talitha L; Kim, Kyungjin; Whitcomb, Daniel J; Collingridge, Graham L; Lightman, Stafford L; Cho, Kwangwook


    The neuroendocrine response to episodes of acute stress is crucial for survival whereas the prolonged response to chronic stress can be detrimental. Learning and memory are particularly susceptible to stress with cognitive deficits being well characterized consequences of chronic stress. Although there is good evidence that acute stress can enhance cognitive performance, the mechanism(s) for this are unclear. We find that hippocampal slices, either prepared from rats following 30 min restraint stress or directly exposed to glucocorticoids, exhibit an N-methyl-d-aspartic acid receptor-independent form of long-term potentiation. We demonstrate that the mechanism involves an NMDA receptor and PKA-dependent insertion of Ca2+ -permeable AMPA receptors into synapses. These then trigger the additional NMDA receptor-independent form of LTP during high frequency stimulation.

  8. Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

    DEFF Research Database (Denmark)

    Vogensen, S B; Jensen, H S; Stensbøl, T B;


    tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50......) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed...

  9. Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit counting (United States)

    Durisic, Nela; Godin, Antoine G.; Wever, Claudia M.; Heyes, Colin D.; Lakadamyali, Melike; Dent, Joseph A.


    The subunit stoichiometry of heteromeric glycine-gated channels (GlyRs) determines fundamental properties of these key inhibitory neurotransmitter receptors; however the ratio of α1 to β-subunits per receptor remains controversial. We used single molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations. PMID:22973015

  10. The antidepressant-like effects of glutamatergic drugs ketamine and AMPA receptor potentiator LY 451646 are preserved in bdnf⁺/⁻ heterozygous null mice. (United States)

    Lindholm, Jesse S O; Autio, Henri; Vesa, Liisa; Antila, Hanna; Lindemann, Lothar; Hoener, Marius C; Skolnick, Phil; Rantamäki, Tomi; Castrén, Eero


    Accumulating evidence suggests that biogenic amine-based antidepressants act, at least in part, via regulation of brain-derived neurotrophic factor (BDNF) signaling. Biogenic amine-based antidepressants increase BDNF synthesis and activate its signaling pathway through TrkB receptors. Moreover, the antidepressant-like effects of these molecules are abolished in BDNF deficient mice. Glutamate-based drugs, including the NMDA antagonist ketamine, and the AMPA receptor potentiator LY 451646, mimic the effects of antidepressants in preclinical tests with high predictive validity. In humans, a single intravenous dose of ketamine produces an antidepressant effect that is rapid, robust and persistent. In this study, we examined the role of BDNF in expression of the antidepressant-like effects of ketamine and an AMPA receptor potentiator (LY 451646) in the forced swim test (FST). Ketamine and LY 451646 produced antidepressant-like effects in the FST in mice at 45 min after a single injection, but no effects were observed one week after a single ketamine injection. As previously reported, the effects of imipramine in the forced swim test were blunted in heterozygous BDNF knockout (bdnf(+/-)) mice. However ketamine and LY 451646 produced similar antidepressant-like responses in wildtype and bdnf(+/-) mice. Neither ketamine nor LY 451646 significantly influenced the levels BDNF or TrkB phosphorylation in the hippocampus when assessed at 45 min or 7 days after the drug administration. These data demonstrate that under the conditions tested, neither ketamine nor the AMPA-potentiator LY 451656 activate BDNF signaling, but produce a characteristic antidepressant-like response in heterozygous bdnf(+/-) mice. These data indicate that unlike biogenic amine-based agents, BDNF signaling does not play a pivotal role in the antidepressant effects of glutamate-based compounds. This article is part of a Special Issue entitled 'Anxiety and Depression'.

  11. Efecto neuroprotector de los cannabinoides sobre la muerte neuronal inducida por Ampa en la médula espinal: Activación conjunta de los receptores CB1 y CB2

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    Carmen Guaza


    Full Text Available La sobreactivación de receptores de glutamato, como el receptor AMPA, induce la muerte neural por un proceso denominado excitotoxicidad, el cual ha sido claramente implicado en enfermedades agudas del sistema nerviso central (SNC, particularmente con daño axonal.

  12. A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression.

    Directory of Open Access Journals (Sweden)

    Andrew R Gallimore


    Full Text Available The expression of long-term depression (LTD in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins-glutamate receptor-interacting protein (GRIP and protein interacting with C kinase 1 (PICK1-regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process.

  13. Calcium-permeable AMPA receptors in the VTA and nucleus accumbens after cocaine exposure: When, how and why?

    Directory of Open Access Journals (Sweden)

    Marina E Wolf


    Full Text Available In animal models of drug addiction, cocaine exposure has been shown to increase levels of calcium-permeable AMPA receptors (CP-AMPARs in two brain regions that are critical for motivation and reward - the ventral tegmental area (VTA and the nucleus accumbens (NAc. This review compares CP-AMPAR plasticity in the two brain regions and addresses its functional significance. In VTA dopamine neurons, cocaine exposure results in synaptic insertion of high conductance CP-AMPARs in exchange for lower conductance calcium-impermeable AMPARs (CI-AMPARs. This plasticity is rapid (hours, GluA2-dependent, and can be observed with a single cocaine injection. In addition to strengthening synapses and altering Ca2+ signaling, CP-AMPAR insertion affects subsequent induction of plasticity at VTA synapses. However, CP-AMPAR insertion is unlikely to mediate the increased dopamine cell activity that occurs during early withdrawal from cocaine exposure. Within the VTA, the group I metabotropic glutamate receptor mGluR1 exerts a negative influence on CP-AMPAR accumulation. Acutely, mGluR1 stimulation elicits a form of LTD resulting from CP-AMPAR removal and CI-AMPAR insertion. In medium spiny neurons (MSNs of the NAc, extended access cocaine self-administration is required to increase CP-AMPAR levels. This is first detected after approximately a month of withdrawal and then persists. Once present in NAc synapses, CP-AMPARs mediate the expression of incubation of cue-induced cocaine craving. The mechanism of their accumulation may be GluA1-dependent, which differs from that observed in the VTA. However, similar to VTA, mGluR1 stimulation removes CP-AMPARs from MSN synapses. Loss of mGluR1 tone during cocaine withdrawal may contribute to CP-AMPAR accumulation in the NAc. Thus, results in both brain regions point to the possibility of using positive modulators of mGluR1 as a treatment for cocaine addiction.

  14. Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor. (United States)

    McQuade, R; Thomas, C G; Nayfeh, S N


    The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves' disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains

  15. Selective Pyramidal Cell Reduction of GABAA Receptor α1 Subunit Messenger RNA Expression in Schizophrenia


    Glausier, Jill R; Lewis, David A.


    Levels of messenger RNA (mRNA) for the α1 subunit of the GABAA receptor, which is present in 60% of cortical GABAA receptors, have been reported to be lower in layer 3 of the prefrontal cortex (PFC) in subjects with schizophrenia. This subunit is expressed in both pyramidal cells and interneurons, and thus lower α1 subunit levels in each cell population would have opposite effects on net cortical excitation. We used dual-label in situ hybridization to quantify GABAA α1 subunit mRNA expression...

  16. Regulation of AMPA and NMDA receptor-mediated EPSPs in dendritic trees of thalamocortical cells. (United States)

    Lajeunesse, Francis; Kröger, Helmut; Timofeev, Igor


    Two main excitatory synapses are formed at the dendritic arbor of first-order nuclei thalamocortical (TC) neurons. Ascending sensory axons primarily establish contacts at large proximal dendrites, whereas descending corticothalamic fibers form synapses on thin distal dendrites. With the use of a multicomparment computational model based on fully reconstructed TC neurons from the ventroposterolateral nucleus of the cat, we compared local responses at the site of stimulation as well as somatic responses induced by both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- and N-methyl-D-aspartate receptor (NMDAR)-mediated currents. We found that AMPAR-mediated responses, when synapses were located at proximal dendrites, induced a larger depolarization at the level of soma, whereas NMDAR-mediated responses were more efficient for synapses located at distal dendrites. The voltage transfer and transfer impedance were higher for NMDAR than for AMPAR activation at any location. For both types of synaptic current and for both input locations at the dendritic arbor, somatic responses were characterized by a low variability despite the large variability found in local responses in dendrites. The large neurons had overall smaller somatic responses than small neurons, but this relation was not found in local dendritic responses. We conclude that in TC cells, the dendritic location of small synaptic inputs does not play a major role in the amplitude of a somatic response, but the size of the neuron does. The variability of response amplitude between cells was much larger than the variability within cells. This suggests possible functional segregation of TC neurons of different size.

  17. Glutamate receptor subunit 3 (GluR3) immunoreactivity delineates a subpopulation of parvalbumin-containing interneurons in the rat hippocampus. (United States)

    Moga, Diana E; Janssen, William G M; Vissavajjhala, Prabhakar; Czelusniak, Sharon M; Moran, Thomas M; Hof, Patrick R; Morrison, John H


    Rasmussen's encephalitis is a childhood disease resulting in intractable seizures associated with hippocampal and neocortical inflammation. An autoantibody against the GluR3 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors is implicated in the pathophysiology of Rasmussen's encephalitis. AMPA receptors mediate excitatory neurotransmission in the brain and contain combinations of four subunits (GluR1-4). Although the distributions of GluR1, GluR2, and GluR4 are known in some detail, the cellular distribution of GluR3 in the mammalian brain remains to be described. We developed and characterized a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in CA1 of the rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and astrocytes and in most interneurons. We quantified the intensity of GluR3 immunoreactivity in interneuron subtypes defined by their calcium-binding protein content. GluR3 immunofluorescence, but not GluR1 or GluR2 immunofluorescence, was significantly elevated in somata of parvalbumin-containing interneurons compared to pyramidal somata. Strikingly, increased GluR3 immunofluorescence was not observed in calbindin- and calretinin-containing interneurons. Furthermore, 24% of parvalbumin-containing interneurons could be distinguished from surrounding neurons based on their intense GluR3 immunoreactivity. This subpopulation had significantly elevated GluR3 immunoreactivity compared to the rest of parvalbumin-containing interneurons. Electron microscopy revealed enriched GluR3 immunoreactivity in parvalbumin-containing perikarya at cytoplasmic and postsynaptic sites. Parvalbumin-containing interneurons, potent inhibitors of cortical pyramidal neurons, are vulnerable in the brains of epileptic patients. Our findings suggest that the somata of these interneurons are enriched in GluR3, which may render them vulnerable to pathological states such as epilepsy and

  18. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David


    Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

  19. A role for calcium-permeable AMPA receptors in synaptic plasticity and learning.

    Directory of Open Access Journals (Sweden)

    Brian J Wiltgen

    Full Text Available A central concept in the field of learning and memory is that NMDARs are essential for synaptic plasticity and memory formation. Surprisingly then, multiple studies have found that behavioral experience can reduce or eliminate the contribution of these receptors to learning. The cellular mechanisms that mediate learning in the absence of NMDAR activation are currently unknown. To address this issue, we examined the contribution of Ca(2+-permeable AMPARs to learning and plasticity in the hippocampus. Mutant mice were engineered with a conditional genetic deletion of GluR2 in the CA1 region of the hippocampus (GluR2-cKO mice. Electrophysiology experiments in these animals revealed a novel form of long-term potentiation (LTP that was independent of NMDARs and mediated by GluR2-lacking Ca(2+-permeable AMPARs. Behavioral analyses found that GluR2-cKO mice were impaired on multiple hippocampus-dependent learning tasks that required NMDAR activation. This suggests that AMPAR-mediated LTP interferes with NMDAR-dependent plasticity. In contrast, NMDAR-independent learning was normal in knockout mice and required the activation of Ca(2+-permeable AMPARs. These results suggest that GluR2-lacking AMPARs play a functional and previously unidentified role in learning; they appear to mediate changes in synaptic strength that occur after plasticity has been established by NMDARs.

  20. Orchestrated regulation of Nogo receptors, LOTUS, AMPA receptors and BDNF in an ECT model suggests opening and closure of a window of synaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Max Nordgren

    Full Text Available Electroconvulsive therapy (ECT is an efficient and relatively fast acting treatment for depression. However, one severe side effect of the treatment is retrograde amnesia, which in certain cases can be long-term. The mechanisms behind the antidepressant effect and the amnesia are not well understood. We hypothesized that ECT causes transient downregulation of key molecules needed to stabilize synaptic structure and to prevent Ca2+ influx, and a simultaneous increase in neurotrophic factors, thus providing a short time window of increased structural synaptic plasticity. Here we followed regulation of NgR1, NgR3, LOTUS, BDNF, and AMPA subunits GluR1 and GluR2 flip and flop mRNA levels in hippocampus at 2, 4, 12, 24, and 72 hours after a single episode of induced electroconvulsive seizures (ECS in rats. NgR1 and LOTUS mRNA levels were transiently downregulated in the dentate gyrus 2, 4, 12 and 4, 12, 24 h after ECS treatment, respectively. GluR2 flip, flop and GluR1 flop were downregulated at 4 h. GluR2 flip remained downregulated at 12 h. In contrast, BDNF, NgR3 and GluR1 flip mRNA levels were upregulated. Thus, ECS treatment induces a transient regulation of factors important for neuronal plasticity. Our data provide correlations between ECS treatment and molecular events compatible with the hypothesis that both effects and side effects of ECT may be caused by structural synaptic rearrangements.

  1. Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression. (United States)

    McCartney, Amber J; Zolov, Sergey N; Kauffman, Emily J; Zhang, Yanling; Strunk, Bethany S; Weisman, Lois S; Sutton, Michael A


    Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

  2. Blocking GABA(A) inhibition reveals AMPA- and NMDA-receptor-mediated polysynaptic responses in the CA1 region of the rat hippocampus. (United States)

    Crépel, V; Khazipov, R; Ben-Ari, Y


    We have investigated the conditions required to evoke polysynaptic responses in the isolated CA1 region of hippocampal slices from Wistar adult rats. Experiments were performed with extracellular and whole cell recording techniques. In the presence of bicuculline (10 microM), 6-cyano-7-nitroquinoxaline-2-3-dione (10 microM), glycine (10 microM), and a low external concentration of Mg2+ (0.3 mM), electrical stimulation of the Schaffer collaterals/commissural pathway evoked graded N-methyl-D-aspartate (NMDA)-receptor-mediated late field potentials in the stratum radiatum of the CA1 region. These responses were generated via polysynaptic connections because their latency varied strongly and inversely with the stimulation intensity and they were abolished by a high concentration of divalent cations (7 mM Ca2+). These responses likely were driven by local collateral branches of CA1 pyramidal cell axons because focal application of tetrodotoxin (30 microM) in the stratum oriens strongly reduced the late synaptic component and antidromic stimulation of CA1 pyramidal cells could evoke the polysynaptic response. Current-source density analysis suggested that the polysynaptic response was generated along the proximal part of the apical dendrites of CA1 pyramidal cells (50-150 microm below the pyramidal cell layer in the stratum radiatum). In physiological concentration of Mg2+ (1.3 mM), the pharmacologically isolated NMDA-receptor-mediated polysynaptic response was abolished. In control artificial cerebrospinal fluid (with physiological concentration of Mg2+), bicuculline ( 10 microM) generated a graded polysynaptic response. Under these conditions, this response was mediated both by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/NMDA receptors. In the presence of D-2-amino-5-phosphonovalerate (50 microM), the polysynaptic response could be mediated by AMPA receptors, although less efficiently. In conclusion, suppression of gamma-aminobutyric acid

  3. Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior. (United States)

    Choi, Jeong Il; Svensson, Camilla I; Koehrn, Fred J; Bhuskute, Aditi; Sorkin, Linda S


    In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca(2+) permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post-treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100microg). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3-kinase (PI-3K) (wortmannin; 1 and 5microg) and LY294002; 50 and 100microg) and Akt (Akt inhibitor IV; 3microg). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and alpha-motor neurons (peak 45min) and later (peak 2h post-injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF-mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF-mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.

  4. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis;


    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep...

  5. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit (United States)

    Martínez-Torres, Ataúlfo; Miledi, Ricardo


    γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

  6. Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons. (United States)

    Zhang, Wen-Bo; Ross, P Joel; Tu, YuShan; Wang, Yongqian; Beggs, Simon; Sengar, Ameet S; Ellis, James; Salter, Michael W


    NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39-57), but not a Src-interfering peptide, Src(40-58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.

  7. Ca2+ imaging of mouse neocortical interneurone dendrites: Contribution of Ca2+-permeable AMPA and NMDA receptors to subthreshold Ca2+dynamics (United States)

    Goldberg, Jesse H; Yuste, Rafael; Tamas, Gabor


    In this second study, we have combined two-photon calcium imaging with whole-cell recording and anatomic reconstructions to directly characterize synaptically evoked calcium signals in three types of mouse V1 supragranular interneurones: parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells (AD). We observed that subthreshold synaptic activation evoked calcium signals locally restricted to individual dendritic compartments. These signals were mediated by NMDA receptors (NMDARs) in AD and IS cells, whereas in FS cells, calcium-permeable AMPA receptors (CP-AMPARs) provided an additional and kinetically distinct influx. Furthermore, even a single, subthreshold synaptic activation evoked a larger dendritic calcium influx than backpropagating action potentials. Our results demonstrate that NMDARs dominate subthreshold calcium dynamics in interneurones and reveal the functional contribution of CP-AMPARs to a specific subclass of cortical interneurone. These data highlight different strategies in dendritic signal processing by distinct classes of interneurones. PMID:12844507

  8. Effect of curcumin on AMPA and kainate receptor-mediated calcium influx in cultured rat hippocampal neurons%姜黄素对AMPA/KA受体介导大鼠海马神经元钙内流的影响

    Institute of Scientific and Technical Information of China (English)

    杜鹏; 彭伟锋; 刘剑英; 林豪杰; 马昱; 汪昕; 范薇


    目的 探讨姜黄素对α-氨基-3-羧基-5-甲基异恶唑-4-丙酸(AMPA)/海人酸(KA)受体介导大鼠海马神经元钙内流的影响.方法 选用胚胎17dSD鼠分离海马,离体培养海马神经元,借助活体钙荧光染色和激光共聚焦钙成像技术观察100μmol/LKA刺激海马神经元内钙的变化,不同浓度(5、10、15、30、50 μmol/L)姜黄素预孵育海马神经元30min对100μmol/L KA刺激下细胞内钙变化的影响,15 μmol/L姜黄素对不同浓度(10、30、50、100、200、300 μmol/L)KA刺激海马神经元内钙变化的影响.应用钴染色技术观察(30、100 μmol/L KA)刺激后海马神经元钴阳性染色细胞变化.姜黄素预孵育30min对KA刺激导致钴阳性染色细胞变化的影响.结果 不同浓度姜黄素预孵育30 min均可以明显缓解100 μmol/L或30 μmol/L KA导致的细胞内钙升高程度.差异均有统计学意义(P<0.05),其中15 μmol/L姜黄素作用最为明显.30μmol/L或100 μmol/LKA刺激均可以引起海马神经元钴染色阳性细胞增加,15 μmol/L姜黄素预处理30 min后明显减少钴染色阳性细胞,差异有统计学意义(P<0.05),而其他浓度(5 μmol/L或30 μmol/L)姜黄素未见明显影响.结论 一定浓度的姜黄素可以影响AMPA/KA受体介导大鼠海马神经元钙内流.这可能是姜黄素抗癫痫作用的一个机制.%Objective To investigate the effect of curcumin on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) receptor-mediated calcium influx in cultured rat hippocampal neurons. Methods The hippocampal neurons from SD rat embryos (17 days old) were cultured for 9 days, and fluorescent calcium chelator and confocal microscopy calcium imaging were used to observe the changes in intracellular free calcium in the neurons following stimulation with 100 μmol/L KA. The effect of curcumin pretreatment at different concentrations (10, 30, 50, 100, 200 and 300 μmol/L) for 30 min on 100 μmol/L KA

  9. Characterization of the 1H-cyclopentapyrimidine-2,4(1H,3H)-dione derivative (S)-CPW399 as a novel, potent, and subtype-selective AMPA receptor full agonist with partial desensitization properties

    DEFF Research Database (Denmark)

    Campiani, G; Morelli, E; Nacci, V;


    (S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound...

  10. Phenylthiophenecarboxamide antagonists of the olfactory receptor co-receptor subunit from a mosquito.

    Directory of Open Access Journals (Sweden)

    Sisi Chen

    Full Text Available Insects detect environmental chemicals using chemosensory receptors, such as the ORs, a family of odorant-gated ion channels. Insect ORs are multimeric complexes of unknown stoichiometry, formed by a common subunit (the odorant receptor co-receptor subunit, Orco and one of many variable subunits that confer odorant specificity. The recent discovery of Orco directed ligands, including both agonists and antagonists, suggests Orco as a promising target for chemical control of insects. In addition to competitively inhibiting OR activation by Orco agonists, several Orco antagonists have been shown to act through a non-competitive mechanism to inhibit OR activation by odorants. We previously identified a series of Orco antagonists, including N-(4-ethylphenyl-2-thiophenecarboxamide (OX1a, previously referred to as OLC20. Here, we explore the chemical space around the OX1a structure to identify more potent Orco antagonists. Cqui\\Orco+Cqui\\Or21, an OR from Culex quinquefasciatus (the Southern House Mosquito that responds to 3-methylindole (skatole and is thought to mediate oviposition behavior, was expressed in Xenopus oocytes and receptor function assayed by two-electrode voltage clamp electrophysiology. 22 structural analogs of OX1a were screened for antagonism of OR activation by an Orco agonist. By varying the moieties decorating the phenyl and thiophene rings, and altering the distance between the rings, we were able to identify antagonists with improved potency. Detailed examination of three of these compounds (N-mesityl-2-thiophenecarboxamide, N-(4-methylbenzyl-2-thiophenecarboxamide and N-(2-ethylphenyl-3-(2-thienyl-2-propenamide demonstrated competitive inhibition of receptor activation by an Orco agonist and non-competitive inhibition of receptor activation by an odorant. The ability to inhibit OR activation by odorants may be a general property of this class of Orco antagonist, suggesting that odorant mediated behaviors can be manipulated

  11. AMPA Receptor-mTOR Activation Is Required for the Antidepressant-like Effects of Sarcosine during the Forced Swim Test in rats: Insertion of AMPA Receptor may Play a Role

    Directory of Open Access Journals (Sweden)

    Kuang-Ti eChen


    Full Text Available Sarcosine, an endogenous amino acid, is a competitive inhibitor of the type I glycine transporter and an N-methyl-D-aspartate receptor (NMDAR coagonist. Recently, we found that sarcosine, an NMDAR enhancer, can improve depression-related behaviors in rodents and humans. This result differs from previous studies, which have reported antidepressant effects of NMDAR antagonists. The mechanisms underlying the therapeutic response of sarcosine remain unknown. This study examines the role of mammalian target of rapamycin (mTOR signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR activation, which are involved in the antidepressant-like effects of several glutamatergic system modulators. The effects of sarcosine in a forced swim test (FST and the expression levels of phosphorylated mTOR signaling proteins were examined in the absence or presence of mTOR and AMPAR inhibitors. In addition, the influence of sarcosine on AMPAR trafficking was determined by analyzing the phosphorylation of AMPAR subunit GluR1 at the PKA site (often considered an indicator for GluR1 membrane insertion in neurons. A single injection of sarcosine exhibited antidepressant-like effects in rats in the FST and rapidly activated the mTOR signaling pathway, which were significantly blocked by mTOR inhibitor rapamycin or the AMPAR inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(fquinoxaline (NBQX pretreatment. Moreover, NBQX pretreatment eliminated the ability of sarcosine to stimulate the phosphorylated mTOR signaling proteins. Furthermore, GluR1 phosphorylation at its PKA site was significantly increased after an acute in vivo sarcosine treatment. The results demonstrated that sarcosine exerts antidepressant-like effects by enhancing AMPAR–mTOR signaling pathway activity and facilitating AMPAR membrane insertion.Highlights:- A single injection of sarcosine rapidly exerted antidepressant-like effects with a concomitant increase in the activation of the

  12. Role of GluR2 expression in AMPA-induced toxicity in cultured murine cerebral cortical neurons

    DEFF Research Database (Denmark)

    Jensen, J B; Lund, Trine Meldgaard; Timmermann, D B;


    of the Mg(2+) block of the NMDA receptor on AMPA-R stimulation. The involvement of Ca(2+) influx through AMPA-R was also examined. The number of neurons possessing Ca(2+)-permeable AMPA-R increased during culture development, concurrently with an increasing susceptibility for AMPA-induced toxicity during...

  13. Episodic sucrose intake during food restriction increases synaptic abundance of AMPA receptors in nucleus accumbens and augments intake of sucrose following restoration of ad libitum feeding. (United States)

    Peng, X-X; Lister, A; Rabinowitsch, A; Kolaric, R; Cabeza de Vaca, S; Ziff, E B; Carr, K D


    Weight-loss dieting often leads to loss of control, rebound weight gain, and is a risk factor for binge pathology. Based on findings that food restriction (FR) upregulates sucrose-induced trafficking of glutamatergic AMPA receptors to the nucleus accumbens (NAc) postsynaptic density (PSD), this study was an initial test of the hypothesis that episodic "breakthrough" intake of forbidden food during dieting interacts with upregulated mechanisms of synaptic plasticity to increase reward-driven feeding. Ad libitum (AL) fed and FR subjects consumed a limited amount of 10% sucrose, or had access to water, every other day for 10 occasions. Beginning three weeks after return of FR rats to AL feeding, when 24-h chow intake and rate of body weight gain had normalized, subjects with a history of sucrose intake during FR consumed more sucrose during a four week intermittent access protocol than the two AL groups and the group that had access to water during FR. In an experiment that substituted noncontingent administration of d-amphetamine for sucrose, FR subjects displayed an enhanced locomotor response during active FR but a blunted response, relative to AL subjects, during recovery from FR. This result suggests that the enduring increase in sucrose consumption is unlikely to be explained by residual enhancing effects of FR on dopamine signaling. In a biochemical experiment which paralleled the sucrose behavioral experiment, rats with a history of sucrose intake during FR displayed increased abundance of pSer845-GluA1, GluA2, and GluA3 in the NAc PSD relative to rats with a history of FR without sucrose access and rats that had been AL throughout, whether they had a history of episodic sucrose intake or not. A history of FR, with or without a history of sucrose intake, was associated with increased abundance of GluA1. A terminal 15-min bout of sucrose intake produced a further increase in pSer845-GluA1 and GluA2 in subjects with a history of sucrose intake during FR

  14. Decreased AMPA GluR2, but not GluR3, mRNA expression in rat amygdala and dorsal hippocampus following morphine-induced behavioural sensitization. (United States)

    Sepehrizadeh, Zargham; Bahrololoumi Shapourabadi, Mina; Ahmadi, Shamseddin; Hashemi Bozchlou, Saeed; Zarrindast, Mohammad-Reza; Sahebgharani, Mousa


    1. Repeated administration of psychostimulants and micro-opioid receptor agonists elicits a progressive enhancement of drug-induced behavioural responses, a phenomenon termed behavioural sensitization. These changes in behaviour may reflect plastic changes requiring regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor function. 2. In the present study, rats were treated for 7 days with saline or morphine (10 mg/kg). After a washout period of either 24 h or 7 days, locomotion, oral stereotypy and state-dependent memory in a passive avoidance test were measured in the presence or absence of 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX; 3 mg/kg), an AMPA receptor antagonist. In order to evaluate the mechanism underlying the behavioural responses, quantitative real-time reverse transcription-polymerase chain reaction was used to evaluate mRNA expression of the AMPA receptor subunits GluR2 and GluR3 in the striatum, prefrontal cortex, hippocampus, hypothalamus and amygdala of animals treated repeatedly with morphine. 3. The results indicate that repeated morphine treatment followed by 7 days (but not 24 h) washout produces behavioural sensitization, as determined by locomotion, oral stereotypy and state-dependent memory. Blockade of AMPA receptors with CNQX on the test day did not alter these behavioural responses. In addition, repeated morphine treatment followed by 7 days (but not 24 h) washout decreased GluR2 mRNA expression in both the amygdala (by 50%) and hippocampus (by 35%). Repeated morphine treatment did not alter GluR3 mRNA expression in any brain area assessed. 4. These data imply that AMPA receptors are involved in the development (but not expression) phase of behavioural sensitization. The decreases in GluR2 mRNA expression in the amygdala and hippocampus may result in the formation of calcium-permeable AMPA receptors, which are believed to play an important role in behavioural sensitization.

  15. AMPA Receptor-mTOR Activation is Required for the Antidepressant-Like Effects of Sarcosine during the Forced Swim Test in Rats: Insertion of AMPA Receptor may Play a Role. (United States)

    Chen, Kuang-Ti; Tsai, Mang-Hung; Wu, Ching-Hsiang; Jou, Ming-Jia; Wei, I-Hua; Huang, Chih-Chia


    Sarcosine, an endogenous amino acid, is a competitive inhibitor of the type I glycine transporter and an N-methyl-d-aspartate receptor (NMDAR) coagonist. Recently, we found that sarcosine, an NMDAR enhancer, can improve depression-related behaviors in rodents and humans. This result differs from previous studies, which have reported antidepressant effects of NMDAR antagonists. The mechanisms underlying the therapeutic response of sarcosine remain unknown. This study examines the role of mammalian target of rapamycin (mTOR) signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) activation, which are involved in the antidepressant-like effects of several glutamatergic system modulators. The effects of sarcosine in a forced swim test (FST) and the expression levels of phosphorylated mTOR signaling proteins were examined in the absence or presence of mTOR and AMPAR inhibitors. In addition, the influence of sarcosine on AMPAR trafficking was determined by analyzing the phosphorylation of AMPAR subunit GluR1 at the PKA site (often considered an indicator for GluR1 membrane insertion in neurons). A single injection of sarcosine exhibited antidepressant-like effects in rats in the FST and rapidly activated the mTOR signaling pathway, which were significantly blocked by mTOR inhibitor rapamycin or the AMPAR inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) pretreatment. Moreover, NBQX pretreatment eliminated the ability of sarcosine to stimulate the phosphorylated mTOR signaling proteins. Furthermore, GluR1 phosphorylation at its PKA site was significantly increased after an acute in vivo sarcosine treatment. The results demonstrated that sarcosine exerts antidepressant-like effects by enhancing AMPAR-mTOR signaling pathway activity and facilitating AMPAR membrane insertion. Highlights-A single injection of sarcosine rapidly exerted antidepressant-like effects with a concomitant increase in the activation of the mammalian

  16. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne


    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  17. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  18. Α2 GABAA receptor sub-units in the ventral hippocampus and α5 GABAA receptor sub-units in the dorsal hippocampus mediate anxiety and fear memory. (United States)

    McEown, K; Treit, D


    Temporary neuronal inactivation of the ventral hippocampus with the GABAA agonist muscimol suppresses unconditioned fear behavior (anxiety) but inactivation of the dorsal hippocampus does not. On the other hand, inactivating the dorsal hippocampus disrupts fear memory, while inactivating the ventral hippocampus does not. Here we investigate the roles of hippocampal GABAA receptor sub-units in mediating these anxiolytic and amnesic effects of GABAA receptor agonists. We microinfused TPA023 (α2 agonist) or TB-21007 (inverse α5 agonist) into the dorsal or ventral hippocampus prior to testing rats in two animal models of anxiety: the elevated plus-maze and shock-probe burying test. Twenty-four hours later rats were re-tested in the shock-probe chamber with a non-electrified probe to assess their memory of the initial shock-probe experience (i.e., fear memory). We found that TPA023 was anxiolytic in the plus-maze and shock-probe burying tests when microinfused into the ventral hippocampus. However, TPA023 did not affect anxiety-related behavior when infused into the dorsal hippocampus. Conversely, we found that the α5 sub-unit inverse agonist TB-21007 impaired rats' memory of the initial shock-probe experience when infused into the dorsal hippocampus, but not when infused into the ventral hippocampus. This double dissociation suggests that α2 GABAA receptor sub-units in the ventral hippocampus mediate unconditioned fear or anxiety, while α5 GABAA receptor sub-units in the dorsal hippocampus mediate conditioned fear memory.

  19. GABAA receptor β3 subunit expression regulates tonic current in developing striatopallidal medium spiny neurons

    Directory of Open Access Journals (Sweden)

    Megan eJanssen


    Full Text Available The striatum is a key structure for movement control, but the mechanisms that dictate the output of distinct subpopulations of medium spiny projection neurons (MSNs, striatonigral projecting and dopamine D1 receptor- (D1+ or striatopallidal projecting and dopamine D2 receptor- (D2+ expressing neurons, remains poorly understood. GABA-mediated tonic inhibition largely controls neuronal excitability and action potential firing rates, and we previously suggested with pharmacological analysis that the GABAA receptor β3 subunit plays a large role in the basal tonic current seen in D2+ MSNs from young mice (Ade et al, 2008; Janssen et al, 2009. In this study, we demonstrated the essential role of the β3 GABAA receptor subunit in mediating MSN tonic currents using conditional β3 subunit knock-out (β3f/fDrd2 mice. Cre-lox genetics were used to generate conditional knock-out animals where Cre recombinase was expressed under the D2 receptor (Drd2 promoter. We show that while the wild-type MSN tonic current pattern demonstrates a high degree of variability, tonic current patterns from β3f/fDrd2 mice are narrow, suggesting that the β3 subunit is essential to striatal MSN GABA-mediated tonic current. Our data also suggest that a distinct population of synaptic receptors upregulate due to β3 subunit removal. Further, deletion of this subunit significantly decreases the D2+ MSN excitability. These results offer insight for target mechanisms in Parkinson’s disease, where symptoms arise due to the imbalance in striatal D1+ and D2+ MSN excitability and output.

  20. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls (United States)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)


    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  1. Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors. (United States)

    Kowalski, Maria; Hausmann, Ralf; Schmid, Julia; Dopychai, Anke; Stephan, Gabriele; Tang, Yong; Schmalzing, Günther; Illes, Peter; Rubini, Patrizia


    The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wild type P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration-response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 receptors. Whereas the allosteric modulators H+ and Zn2+ failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of P2X2 and P2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP)2/(P2X3)1 complex and a (P2X2-GFP)1/(P2X3)2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits.

  2. Mouse Leydig cells express multiple P2X receptor subunits



    ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X1–P2X7) and seven heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/6, P2X4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca2+]i and testosterone secretion, supporting the hypothesis that Ca2+ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Ley...

  3. Insights into the mechanisms of ifosfamide encephalopathy: drug metabolites have agonistic effects on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and induce cellular acidification in mouse cortical neurons. (United States)

    Chatton, J Y; Idle, J R; Vågbø, C B; Magistretti, P J


    Therapeutic value of the alkylating agent ifosfamide has been limited by major side effects including encephalopathy. Although the underlying biochemical processes of the neurotoxic side effects are still unclear, they could be attributed to metabolites rather than to ifosfamide itself. In the present study, the effects of selected ifosfamide metabolites on indices of neuronal activity have been investigated, in particular for S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA). Because of structural similarities of SCMC with glutamate, the Ca(2+)(i) response of single mouse cortical neurons to SCMC and TDGA was investigated. SCMC, but not TDGA, evoked a robust increase in Ca(2+)(i) concentration that could be abolished by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only partly diminished by the N-methyl-D-aspartate receptor antagonist 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK=801). Cyclothiazide (CYZ), used to prevent AMPA/kainate receptor desensitization, potentiated the response to SCMC. Because activation of AMPA/kainate receptors is known to induce proton influx, the intracellular pH (pH(i)) response to SCMC was investigated. SCMC caused a concentration-dependent acidification that was amplified by CYZ. Since H(+)/monocarboxylate transporter (MCT) activity leads to similar cellular acidification, we tested its potential involvement in the pH(i) response. Application of the lactate transport inhibitor quercetin diminished the pH(i) response to SCMC and TDGA by 43 and 51%, respectively, indicating that these compounds may be substrates of MCTs. Taken together, this study indicates that hitherto apparently inert ifosfamide metabolites, in particular SCMC, activate AMPA/kainate receptors and induce cellular acidification. Both processes could provide the biochemical basis of the observed ifosfamide-associated encephalopathy.

  4. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii. (United States)

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo


    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.

  5. SB-205384 Is a Positive Allosteric Modulator of Recombinant GABAA Receptors Containing Rat α3, α5, or α6 Subunit Subtypes Coexpressed with β3 and γ2 Subunits


    Heidelberg, Laura S.; Warren, James W.; Fisher, Janet L.


    Many drugs used to treat anxiety are positive modulators of GABAA receptors, which mediate fast inhibitory neurotransmission. The GABAA receptors can be assembled from a combination of at least 16 different subunits. The receptor’s subunit composition determines its pharmacologic and functional properties, and subunit expression varies throughout the brain. A primary goal for new treatments targeting GABAA receptors is the production of subunit-selective modulators acting upon a discrete popu...

  6. A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy. (United States)

    Löscher, W; Lehmann, H; Behl, B; Seemann, D; Teschendorf, H J; Hofmann, H P; Lubisch, W; Höger, T; Lemaire, H G; Gross, G


    Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-D-aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5-GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA

  7. Inhibin subunits, follistatin and activin receptors in the human teratocarcinoma cell line Tera-2. (United States)

    de Jong, F H; de Winter, J P; Wesseling, J G; Timmerman, M A; van Genesen, S; van den Eijnden-van Raaij, A J; van Zoelen, E J


    The expression of mRNAs for inhibin subunits was studied in the human teratocarcinoma cell line Tera-2 clone 13 before and after differentiation with retinoic acid (RA). Both alpha- and beta B-subunits of inhibin were expressed. Subsequently, inhibin bio- and immunoactivity in the conditioned media of the Tera-2 cells were determined by studying the release of follicle-stimulating hormone from rat pituitary cells, by immunoassay and by immunoprecipitation using inhibin alpha- and beta B-subunit specific antibodies. Strikingly dissimilar high bio- and low immuno-activities were found. The ensuing hypothesis that the high bioactivity might be due to the presence of the activin-binding protein follistatin was confirmed by immunoprecipitation of 34 and 37 kDa labelled proteins, using a polyclonal anti-follistatin antiserum after culture of the Tera-2 cells with [35S]-methionine. Furthermore, expression of activin receptor types II and IIB mRNA was found in the cells. Addition of 5 microM RA to monolayer cultures of Tera-2 cells resulted in differentiation to flat endoderm-like cells and caused a slight suppression of the expression of the mRNA encoding the inhibin alpha- and beta B-subunits. The expression of follistatin and activin receptor type IIB was strongly suppressed, whereas the expression of the activin receptor type II was not affected. We conclude that Tera-2 cells secrete follistatin and express inhibin subunits and activin receptors differentially during RA-induced differentiation. The role of the decreased expression of follistatin and activin receptor IIB mRNA after addition of RA in the mechanism of RA-induced differentiation remains to be elucidated.

  8. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons. (United States)

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo


    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  9. Neonatal Seizures Alter NMDA Glutamate receptor GluN2A and 3A Subunit Expression and Function in Hippocampal CA1 Neurons

    Directory of Open Access Journals (Sweden)

    Chengwen eZhou


    Full Text Available Neonatal seizures are commonly caused by hypoxic and/or ischemic injury during birth and can lead to long-term epilepsy and cognitive deficits. In a rodent hypoxic seizure (HS model, we have previously demonstrated a critical role for seizure-induced enhancement of the AMPA subtype of glutamate receptor (GluA in epileptogenesis and cognitive consequences, in part due to GluA maturational upregulation of expression. Similarly, as the expression and function of the NMDA subtype of glutamate receptor (GluN is also developmentally controlled, we examined how early life seizures during the critical period of synaptogenesis could modify GluN development and function. In a postnatal day (P10 rat model of neonatal seizures, we found that seizures could alter GluN2/3 subunit composition of GluNs and physiological function of synaptic GluNs. In hippocampal slices removed from rats within 48-96 hours following seizures, the amplitudes of synaptic GluN-mediated evoked excitatory postsynaptic currents (eEPSCs were elevated in CA1 pyramidal neurons. Moreover, GluN eEPSCs showed a decreased sensitivity to GluN2B selective antagonists and decreased Mg2+ sensitivity at negative holding potentials, indicating a higher proportion of GluN2A and GluN3A subunit function, respectively. These physiological findings were accompanied by a concurrent increase in GluN2A phosphorylation and GluN3A protein. These results suggest that altered GluN function and expression could potentially contribute to future epileptogenesis following neonatal seizures, and may represent potential therapeutic targets for the blockade of future epileptogenesis in the developing brain.

  10. Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans. (United States)

    Sloan, Megan A; Reaves, Barbara J; Maclean, Mary J; Storey, Bob E; Wolstenholme, Adrian J


    The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing.

  11. Analysis of NR3A receptor subunits in human native NMDA receptors

    DEFF Research Database (Denmark)

    Nilsson, Anna; Eriksson, Maria; Muly, E Chris


    NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human...... primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only...... very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2...

  12. The sigma receptor as a ligand-regulated auxiliary potassium channel subunit. (United States)

    Aydar, Ebru; Palmer, Christopher P; Klyachko, Vitaly A; Jackson, Meyer B


    The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.

  13. Slow-dissociation effect of common signaling subunit beta c on IL5 and GM-CSF receptor assembly. (United States)

    Ishino, Tetsuya; Harrington, Adrian E; Zaks-Zilberman, Meirav; Scibek, Jeffery J; Chaiken, Irwin


    Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit alpha and recruitment of a common signaling subunit beta (betac). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor alpha and betac subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when alpha subunit alone was anchored. The slow-dissociation effect of betac had a similar impact on GM-CSF receptor stabilization to that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in betac, which together constitute key parts of a cytokine binding epitope. The data argue that betac plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of betac explains how, when multiple betac cytokine receptor alpha subunits are present on the same cell surface, selective betac usage can be controlled by sequestration in stabilized cytokine-alpha-betac complexes.

  14. Channel-lining residues of the AMPA receptor M2 segment: structural environment of the Q/R site and identification of the selectivity filter. (United States)

    Kuner, T; Beck, C; Sakmann, B; Seeburg, P H


    In AMPA receptor channels, a single amino acid residue (Q/R site) of the M2 segment controls permeation of calcium ions, single-channel conductance, blockade by intracellular polyamines, and permeation of anions. The structural environment of the Q/R site and its positioning with regard to a narrow constriction were probed with the accessibility of substituted cysteines to positively and negatively charged methanethiosulfonate reagents, applied from the extracellular and cytoplasmic sides of the channel. The accessibility patterns confirm that the M2 segment forms a pore loop with the Q/R site positioned at the tip of the loop (position 0) facing the extracellular vestibule. Cytoplasmically accessible residues on the N- and C-terminal sides of position 0 form the ascending alpha-helical (-8 to -1) and descending random coil (+1 to +6) components of the loop, respectively. Substitution of a glycine residue at position +2 with alanine strongly decreased the permeability of organic cations, indicating that position +2 contributes to the narrow constriction. The anionic 2-sulfonatoethyl-methanethiosufonate reacted with a cysteine at position 0 only from the external side and with cysteines at positions +1 to +4 only from the cytoplasmic side. These results suggest that charge selectivity occurs external to the constriction (+2) and possibly involves interactions of ions with the negative electrostatic potential created by the dipole of the alpha-helix formed by the ascending limb of the loop.

  15. Perampanel: A Selective AMPA Antagonist for Treating Seizures


    Krauss, Gregory L.


    Perampanel is a selective, noncompetitive AMPA receptor antagonist that has recently been approved for treating localization-related epilepsy. This article reviews the pharmacology, clinical development, efficacy, and safety/tolerability of perampanel.

  16. NMDA and non-NMDA receptor gene expression following global brain ischemia in rats: effect of NMDA and non-NMDA receptor antagonists. (United States)

    Pellegrini-Giampietro, D E; Pulsinelli, W A; Zukin, R S


    Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous study, we have shown that expression of GluR2, the kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca2+ permeability, is preferentially reduced in CA1 at a time point preceding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), protects CA1 neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor antagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDAR1. In ischemic rats treated with saline, GluR2 and GluR3 mRNAs were markedly reduced in CA1 but were unchanged in CA3 or dentate gyrus. GluR1 and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modification of GluR2 expression changes.

  17. Design and synthesis of labeled analogs of PhTX-56, a potent and selective AMPA receptor antagonist

    DEFF Research Database (Denmark)

    Andersen, Trine F; Vogensen, Stine B; Jensen, Lars S;


    Polyamines and polyamine toxins are biologically important molecules, having modulatory effects on nucleotides and proteins. The wasp toxin, philanthotoxin-433 (PhTX-433), is a non-selective and uncompetitive antagonist of ionotropic receptors, such as ionotropic glutamate receptors and nicotinic...

  18. Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors

    DEFF Research Database (Denmark)

    Nielsen, B S; Banke, T G; Schousboe, A


    Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after...

  19. The chemokine growth-related gene product β protects rat cerebellar granule cells from apoptotic cell death through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (United States)

    Limatola, Cristina; Ciotti, Maria Teresa; Mercanti, Delio; Vacca, Fabrizio; Ragozzino, Davide; Giovannelli, Aldo; Santoni, Angela; Eusebi, Fabrizio; Miledi, Ricardo


    Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K+. We have found that the growth-related gene product β (GROβ) partially prevents the K+-depletion-induced cell death, and that the neuroprotective action of GROβ on granule cells is mediated through the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GROβ-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-d-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 μM) treatment. Moreover, GROβ-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GROβ is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors. PMID:10811878

  20. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity. (United States)

    Ratra, G S; Kamita, S G; Casida, J E


    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  1. Involvement of AMPA/Kainate Glutamate Receptor in the Extinction and Reinstatement of Morphine-Induced Conditioned Place Preference: A Behavioral and Molecular Study. (United States)

    Siahposht-Khachaki, Ali; Fatahi, Zahra; Yans, Asal; Khodagholi, Fariba; Haghparast, Abbas


    Glutamate receptors in mesolimbic areas such as the nucleus accumbens, ventral tegmental area, prefrontal cortex (PFC), and hippocampus (HIP) are a component of the mechanisms of drug-induced reward and can modulate the firing pattern of dopaminergic neurons in the reward system. In addition, several lines of study have indicated that cAMP response element-binding protein (CREB) and c-fos have important role in morphine-induced conditioned place preference (CPP) induced by drugs of abuse, such as morphine, cocaine, nicotine, and alcohol. Therefore, in the present study, we investigated the changes in phosphorylated CREB (p-CREB) and c-fos induction within the nucleus accumbens (NAc), HIP, and PFC after intracerebroventricular (ICV) administration of different doses of CNQX or vehicle during extinction period or reinstatement of morphine-induced CPP. In all groups, the CPP procedure was done; afterward, the conditioning scores were recorded by Ethovision software. After behavioral test recording, we dissected out the NAc, HIP, and PFC regions and measured the p-CREB/CREB ratio and c-fos level by Western blot analysis. Our results showed that administration of CNQX significantly shortened the extinction of morphine CPP. Besides, ICV microinjection of CNQX following extinction period decreased the reinstatement of morphine CPP in extinguished rats. In molecular section, in treatment group, all mentioned factors were dose-dependently decreased in comparison with vehicle group (DMSO) after ICV microinjection of different doses of CNQX but not in pre-extinction microinjection. These findings suggested that antagonism of AMPA receptor decreased p-CREB/CREB ratio and c-fos level in the PFC, NAc, and HIP. Modulation of the drug memory reconsolidation may be useful for faster extinction of drug-induced reward and attenuation of drug-seeking behavior.

  2. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    Institute of Scientific and Technical Information of China (English)

    Teresa A MURRAY; Qiang LIU; Paul WHITEAKER; Jie WU; Ronald J LUKAS


    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR a.7 subunit without compromising formation of functional receptors.Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR al sub-unit (a7Y). SH-EP1 cells were transfected with mouse nAChR wild type a.7 subunits (a.7) or with a7Y subunits, alone or with the chaperone protein, hRJC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled a-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Results: Whole-cell currents revealed that a7Y nAChRs and al nAChRs were functional with comparable EC50 values for the a7 nAChR-selective agonist, choline, and IC50 values for the a.7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that a7Y had primarily intracel-lular rather than surface expression. TIRF microscopy confirmed that little a7Y localized to the plasma membrane, typical of a7 nAChRs.Conclusion: nAChRs composed as homooligomers of a7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of a.7 nAChRs. a7Y nAChRs may be used to elucidate properties of a.7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

  3. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse (United States)

    Almedom, Ruta B; Liewald, Jana F; Hernando, Guillermina; Schultheis, Christian; Rayes, Diego; Pan, Jie; Schedletzky, Thorsten; Hutter, Harald; Bouzat, Cecilia; Gottschalk, Alexander


    Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the ‘levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. PMID:19609303

  4. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies. (United States)

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen


    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

  5. Molecular pharmacology of the AMPA agonist, (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA] and the AMPA antagonist, (R)-APPA

    DEFF Research Database (Denmark)

    Ebert, B; Madsen, U; Lund, Trine Meldgaard


    The heterocyclic analogue of (S)-glutamic acid, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [(S)-AMPA] is a potent and selective AMPA receptor agonist, whereas the enantiomeric compound, (R)-AMPA, is virtually inactive. We have previously characterized (RS)-2-amino-3-(3-hydroxy-...

  6. Therapeutic window of opportunity for the neuroprotective effect of valproate versus the competitive AMPA receptor antagonist NS1209 following status epilepticus in rats. (United States)

    Langer, Melanie; Brandt, Claudia; Zellinger, Christina; Löscher, Wolfgang


    Epileptogenesis, i.e., the process leading to epilepsy, is a presumed consequence of brain insults including head trauma, stroke, infections, tumors, status epilepticus (SE), and complex febrile seizures. Typically, brain insults produce morphological and functional alterations in the hippocampal formation, including neurodegeneration in CA1, CA3, and, most consistently, the dentate hilus. Most of these alterations develop gradually, over several days, after the insult, providing a therapeutic window of opportunity for neuroprotective agents in the immediate post-injury period. We have previously reported that prolonged (four weeks) treatment with the antiepileptic drug valproate (VPA) after SE prevents hippocampal damage and most of the behavioral alterations that occur after brain insult, but not the development of spontaneously occurring seizures. These data indicated that VPA, although not preventing epilepsy, might be an effective disease-modifying treatment following brain insult. The present study was designed to (1) determine the therapeutic window for the neuroprotective effect of VPA after SE; (2) compare the efficacy of different intermittent i.p. versus continuous i.v. VPA treatment protocols; and (3) compare VPA with the glutamate (AMPA) receptor antagonist NS1209. As in our previous study with VPA, SE was induced by sustained electrical stimulation of the basolateral amygdala in rats and terminated after 4 h by diazepam. In vehicle controls, >90% of the animals developed significant neurodegeneration in the dentate hilus, whereas damage in CA1 and CA3 was more variable. Hilar parvalbumin-expressing interneurons were more sensitive to the effects of seizures than somatostatin-stained hilar interneurons or hilar mossy cells. Among the various VPA treatment protocols, continuous infusion of VPA for 24 immediately following the SE was the most effective neuroprotective treatment, preventing most of the neuronal damage. Infusion with NS1209 for 24 h

  7. Eating 'Junk-Food' Produces Rapid and Long-Lasting Increases in NAc CP-AMPA Receptors: Implications for Enhanced Cue-Induced Motivation and Food Addiction. (United States)

    Oginsky, Max F; Goforth, Paulette B; Nobile, Cameron W; Lopez-Santiago, Luis F; Ferrario, Carrie R


    Urges to eat are influenced by stimuli in the environment that are associated with food (food cues). Obese people are more sensitive to food cues, reporting stronger craving and consuming larger portions after food cue exposure. The nucleus accumbens (NAc) mediates cue-triggered motivational responses, and activations in the NAc triggered by food cues are stronger in people who are susceptible to obesity. This has led to the idea that alterations in NAc function similar to those underlying drug addiction may contribute to obesity, particularly in obesity-susceptible individuals. Motivational responses are mediated in part by NAc AMPA receptor (AMPAR) transmission, and recent work shows that cue-triggered motivation is enhanced in obesity-susceptible rats after 'junk-food' diet consumption. Therefore, here we determined whether NAc AMPAR expression and function is increased by 'junk-food' diet consumption in obesity-susceptible vs -resistant populations using both outbred and selectively bred models of susceptibility. In addition, cocaine-induced locomotor activity was used as a general 'read out' of mesolimbic function after 'junk-food' consumption. We found a sensitized locomotor response to cocaine in rats that gained weight on a 'junk-food' diet, consistent with greater responsivity of mesolimbic circuits in obesity-susceptible groups. In addition, eating 'junk-food' increased NAc calcium-permeable-AMPAR (CP-AMPAR) function only in obesity-susceptible rats. This increase occurred rapidly, persisted for weeks after 'junk-food' consumption ceased, and preceded the development of obesity. These data are considered in light of enhanced cue-triggered motivation and striatal function in obesity-susceptible rats and the role of NAc CP-AMPARs in enhanced motivation and addiction.

  8. Size and receptor density of glutamatergic synapses: a viewpoint from left-right asymmetry of CA3-CA1 connections

    Directory of Open Access Journals (Sweden)

    Yoshiaki Shinohara


    Full Text Available Synaptic plasticity is considered to be the main mechanism for learning and memory. Excitatory synapses in the cerebral cortex and hippocampus undergo plastic changes during development and in response to electric stimulation. It is widely accepted that this process is mediated by insertion and elimination of various glutamate receptors. In a series of recent investigations on left-right asymmetry of hippocampal CA3-CA1 synapses, glutamate receptor subunits have been found to have distinctive expression patterns that depend on the postsynaptic density (PSD area. Particularly notable are the GluR1 AMPA receptor subunit and NR2B NMDA receptor subunit, where receptor density has either a supra-linear (GluR1 AMPA or inverse (NR2B NMDAR relationship to the PSD area. We review current understanding of structural and physiological synaptic plasticity and propose a scheme to classify receptor subtypes by their expression pattern with respect to PSD area.

  9. Mechanism of partial agonism at the GluR2 AMPA receptor: Measurements of lobe orientation in solution. (United States)

    Maltsev, Alexander S; Ahmed, Ahmed H; Fenwick, Michael K; Jane, David E; Oswald, Robert E


    The mechanism by which the binding of a neurotransmitter to a receptor leads to channel opening is a central issue in molecular neurobiology. The structure of the agonist binding domain of ionotropic glutamate receptors has led to an improved understanding of the changes in structure that accompany agonist binding and have provided important clues about the link between these structural changes and channel activation and desensitization. However, because the binding domain has exhibited different structures under different crystallization conditions, understanding the structure in the absence of crystal packing is of considerable importance. The orientation of the two lobes of the binding domain in the presence of a full agonist, an antagonist, and several partial agonists was measured using NMR spectroscopy by employing residual dipolar couplings. For some partial agonists, the solution conformation differs from that observed in the crystal. A model of channel activation based on the results is discussed.

  10. KIS, a kinase associated with microtubule regulators, enhances translation of AMPA receptors and stimulates dendritic spine remodeling. (United States)

    Pedraza, Neus; Ortiz, Raúl; Cornadó, Alba; Llobet, Artur; Aldea, Martí; Gallego, Carme


    Local regulation of protein synthesis allows a neuron to rapidly alter the proteome in response to synaptic signals, an essential mechanism in synaptic plasticity that is altered in many neurological diseases. Synthesis of many synaptic proteins is under local control and much of this regulation occurs through structures termed RNA granules. KIS is a protein kinase that associates with stathmin, a modulator of the tubulin cytoskeleton. Furthermore, KIS is found in RNA granules and stimulates translation driven by the β-actin 3'UTR in neurites. Here we explore the physiological and molecular mechanisms underlying the action of KIS on hippocampal synaptic plasticity in mice. KIS downregulation compromises spine development, alters actin dynamics, and reduces postsynaptic responsiveness. The absence of KIS results in a significant decrease of protein levels of PSD-95, a postsynaptic scaffolding protein, and the AMPAR subunits GluR1 and GluR2 in a CPEB3-dependent manner. Underlying its role in spine maturation, KIS is able to suppress the spine developmental defects caused by CPEB3 overexpression. Moreover, either by direct or indirect mechanisms, KIS counteracts the inhibitory activity of CPEB3 on the GluR2 3'UTR at both mRNA translation and polyadenylation levels. Our study provides insights into the mechanisms that mediate dendritic spine morphogenesis and functional synaptic maturation, and suggests KIS as a link regulating spine cytoskeleton and postsynaptic activity in memory formation.

  11. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms. (United States)

    Li, Ben-Wen; Rush, Amy C; Weil, Gary J


    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of "classical" anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of V as deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects of

  12. Discs-large (DLG is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila

    Directory of Open Access Journals (Sweden)

    Featherstone David E


    Full Text Available Abstract Background Drosophila discs-large (DLG is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors. Results There are thought to be two classes of glutamate receptors in the Drosophila NMJ: 1 receptors that contain the subunit GluRIIA, and 2 receptors that contain the subunit GluRIIB. In DLG mutants, antibody staining for the glutamate receptor subunit GluRIIA is normal, but antibody staining for the glutamate receptor subunit GluRIIB is significantly reduced. Electrophysiological analysis shows an overall loss of functional postsynaptic glutamate receptors, along with changes in receptor biophysical properties that are consistent with a selective loss of GluRIIB from the synapse. In uninnervated postsynaptic muscles, neither glutamate receptors nor DLG cluster at synapses. DLG clusters normally in the complete absence of glutamate receptors. Conclusions Our results suggest that DLG controls glutamate receptor subunit composition by selectively stabilizing GluRIIB-containing receptors at the synapse. We also show that DLG, like glutamate receptors, is localized only after the presynaptic neuron contacts the

  13. NR2 subunits and NMDA receptors on lamina II inhibitory and excitatory interneurons of the mouse dorsal horn

    Directory of Open Access Journals (Sweden)

    MacDermott Amy B


    Full Text Available Abstract Background NMDA receptors expressed by spinal cord neurons in the superficial dorsal horn are involved in the development of chronic pain associated with inflammation and nerve injury. The superficial dorsal horn has a complex and still poorly understood circuitry that is mainly populated by inhibitory and excitatory interneurons. Little is known about how NMDA receptor subunit composition, and therefore pharmacology and voltage dependence, varies with neuronal cell type. NMDA receptors are typically composed of two NR1 subunits and two of four NR2 subunits, NR2A-2D. We took advantage of the differences in Mg2+ sensitivity of the NMDA receptor subtypes together with subtype preferring antagonists to identify the NR2 subunit composition of NMDA receptors expressed on lamina II inhibitory and excitatory interneurons. To distinguish between excitatory and inhibitory interneurons, we used transgenic mice expressing enhanced green fluorescent protein driven by the GAD67 promoter. Results Analysis of conductance ratio and selective antagonists showed that lamina II GABAergic interneurons express both the NR2A/B containing Mg2+ sensitive receptors and the NR2C/D containing NMDA receptors with less Mg2+ sensitivity. In contrast, excitatory lamina II interneurons express primarily NR2A/B containing receptors. Despite this clear difference in NMDA receptor subunit expression in the two neuronal populations, focally stimulated synaptic input is mediated exclusively by NR2A and 2B containing receptors in both neuronal populations. Conclusions Stronger expression of NMDA receptors with NR2C/D subunits by inhibitory interneurons compared to excitatory interneurons may provide a mechanism to selectively increase activity of inhibitory neurons during intense excitatory drive that can provide inhibitory feedback.

  14. Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

    Directory of Open Access Journals (Sweden)

    Torrão A.S.


    Full Text Available Nicotinic acetylcholine receptors (nAChRs are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

  15. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block. (United States)

    Dravid, Shashank M; Erreger, Kevin; Yuan, Hongjie; Nicholson, Katherine; Le, Phuong; Lyuboslavsky, Polina; Almonte, Antoine; Murray, Ernest; Mosely, Cara; Barber, Jeremy; French, Adam; Balster, Robert; Murray, Thomas F; Traynelis, Stephen F


    We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pK(a) values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (-)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.

  16. Positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors reverses subcronic PCP-induced deficits in the novel object recognition task in rats

    DEFF Research Database (Denmark)

    Nielsen, Trine Damgaard; Larsen, Dorrit Bjerg; Hansen, Suzanne Lisbet;


    deficit in female Lister hooded rats in teh novel object recognition (NOR) task. Here we show that positive modulation of AMPA receptor (AMPAR) mediated glutamate transmission alleviates cognitive deficits induced by sub-chronic PCP treatment. Female Lister hooded rats were treated sub......-cbronic PCP treatment induced a significant decrease in the discrimination index (DI) and both ampakines CX546 and CX516 were able to reverse this diruption of object memory in rats in the novel object recognition task. These data suggest that positive AMPAR modulation may represent a mechanism for treatment...

  17. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.


    Pellegrini-Giampietro, D E; Zukin, R.S.; Bennett, M V; Cho, S; Pulsinelli, W. A.


    Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeab...

  18. Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology. (United States)

    Frølund, Bente; Greenwood, Jeremy R; Holm, Mai M; Egebjerg, Jan; Madsen, Ulf; Nielsen, Birgitte; Bräuner-Osborne, Hans; Stensbøl, Tine B; Krogsgaard-Larsen, Povl


    Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.

  19. Evaluation of native GABA(A) receptors containing an alpha 5 subunit. (United States)

    Li, M; Szabo, A; Rosenberg, H C


    The type A receptor for gamma-aminobutyric acid (GABA), or GABA(A) receptor, is a pentamer of highly variable quaternary structure. It includes two alpha subunits, drawn from a pool of six genes, which largely determine benzodiazepine pharmacology of the receptor. In brain sections, both [(3)H]RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate) and [(3)H]L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), which are selective for the benzodiazepine site of alpha 5 subunit-containing receptors, showed high-affinity, specific binding, but to fewer regions than did the nonselective benzodiazepine, [(3)H]flunitrazepam. The pattern mirrored alpha 5 mRNA distribution, and was similar to that previously reported for [(3)H]L-655,708 binding. Displacement of [(3)H]RY-80 bound to hippocampal homogenates, and of [(3)H]flunitrazepam bound to cerebellar and hippocampal homogenates showed comparable displacement by flumazenil (K(i)'s 5--7 nM). However, the K(i)'s for diazepam and for clobazam to displace [(3)H]RY-80 binding in hippocampus were about fourfold higher than for [(3)H]flunitrazepam, and the K(i) for clonazepam was sixfold larger, suggesting that these benzodiazepine receptor agonists bind with relatively lower affinity at hippocampal alpha 5-containing receptors.

  20. Domoic Acid-Induced Neurotoxicity Is Mainly Mediated by the AMPA/KA Receptor: Comparison between Immature and Mature Primary Cultures of Neurons and Glial Cells from Rat Cerebellum

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    Helena T. Hogberg


    Full Text Available Domoic acid (DomA is a naturally occurring shellfish toxin that can induce brain damage in mammalians. Neonates have shown increased sensitivity to DomA-induced toxicity, and prenatal exposure has been associated with e.g. decreased brain GABA levels, and increased glutamate levels. Here, we evaluated DomA-induced toxicity in immature and mature primary cultures of neurons and glial cells from rat cerebellum by measuring the mRNA levels of selected genes. Moreover, we assessed if the induced toxicity was mediated by the activation of the AMPA/KA and/or the NMDA receptor. The expression of all studied neuronal markers was affected after DomA exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment, as the effects were observed at lower concentrations and at earlier time points than for the immature cultures. The DomA effects were completely prevented by the antagonist of the AMPA/KA receptor (NBQX, while the antagonist of the NMDA receptor (APV partly blocked the DomA-induced effects. Interestingly, the DomA-induced effect was also partly prevented by the neurotransmitter GABA. DomA exposure also affected the mRNA levels of the astrocytic markers in mature cultures. These DomA-induced effects were reduced by the addition of NBQX, APV, and GABA.

  1. GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

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    Yuri A Blednov

    Full Text Available GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR, and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist, when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests, but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

  2. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

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    Christine Laura Dixon


    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  3. The GluK4 kainate receptor subunit regulates memory, mood, and excitotoxic neurodegeneration. (United States)

    Lowry, E R; Kruyer, A; Norris, E H; Cederroth, C R; Strickland, S


    Though the GluK4 kainate receptor subunit shows limited homology and a restricted expression pattern relative to other kainate receptor subunits, its ablation results in distinct behavioral and molecular phenotypes. GluK4 knockout mice demonstrated impairments in memory acquisition and recall in a Morris water maze test, suggesting a previously unreported role for kainate receptors in spatial memory. GluK4 knockout mice also showed marked hyperactivity and impaired pre-pulse inhibition, thereby mirroring two of the hallmark endophenotypes of patients with schizophrenia and bipolar disorder. Furthermore, we found that GluK4 is a key mediator of excitotoxic neurodegeneration: GluK4 knockout mice showed robust neuroprotection in the CA3 region of the hippocampus following intrahippocampal injection of kainate and widespread neuroprotection throughout the hippocampus following hypoxia-ischemia. Biochemical analysis of kainate- or sham-treated wild-type and GluK4 knockout hippocampal tissue suggests that GluK4 may act through the JNK pathway to regulate the molecular cascades that lead to excitotoxicity. Together, our findings suggest that GluK4 may be relevant to the understanding and treatment of human neuropsychiatric and neurodegenerative disorders.

  4. The α5 subunit containing GABAA receptors contribute to chronic pain. (United States)

    Bravo-Hernández, Mariana; Corleto, José A; Barragán-Iglesias, Paulino; González-Ramírez, Ricardo; Pineda-Farias, Jorge B; Felix, Ricardo; Calcutt, Nigel A; Delgado-Lezama, Rodolfo; Marsala, Martin; Granados-Soto, Vinicio


    It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15-15 nmol), prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of rate-dependent depression. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.

  5. NMDA receptor NR2B subunits contribute to PTZ-kindling-induced hippocampal astrocytosis and oxidative stress. (United States)

    Zhu, Xinjian; Dong, Jingde; Shen, Kai; Bai, Ying; Zhang, Yuan; Lv, Xuan; Chao, Jie; Yao, Honghong


    The N-methyl-d-aspartate (NMDA) receptor plays an important role in the pathophysiology of several neurological diseases, including epilepsy. The present study investigated the effect of NMDA receptor NR2B subunits on pentylenetetrazole (PTZ)-kindling-induced pathological and biochemical events in mice. Our results showed that PTZ-kindling up-regulates the expression of NMDA receptor NR2B subunits in the hippocampus and that kindled mice were characterized by significant astrocytosis and neuron loss in the hippocampus. Oxidative stress, including excessive malondialdehyde (MDA) production and decreased enzymatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), were detected in the hippocampus after the mice were fully kindled. Additionally, expression of brain-derived neurotrophic factor (BDNF) in the hippocampus was found to be up-regulated in PTZ-kindled mice. However, selectively blocking NMDA receptor NR2B subunits by ifenprodil significantly suppressed PTZ-kindling-induced hippocampal astrocytosis, oxidative stress and neuron loss. Furthermore, blocking NMDA receptor NR2B subunits also abolished PTZ-kindling-induced BDNF expression. These results indicate that NMDA receptor NR2B subunits contribute to epilepsy-associated pathological and biochemical events, including hippocampal astrocytosis, oxidative stress and neuron loss, and these events might be correlated with up-regulation of BDNF expression.

  6. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

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    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  7. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes (United States)

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo


    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  8. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.


    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  9. Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected. (United States)

    Wagoner, Kelly R; Czajkowski, Cynthia


    The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.

  10. Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

    Directory of Open Access Journals (Sweden)

    Gersende Maugars

    Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

  11. Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

    Institute of Scientific and Technical Information of China (English)

    Frank D.Rinkevich; Jeffrey G.Scott


    Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor.The α6 nicotinic acetyl-choline receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6.RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Ga14-UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants.These Dα6-deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity.The expression of Dα6 was reduced 60%-75% as verified by quantitative polymerase chain reaction.However,there was no change in spinosad sensitivity in D.melanogaster.We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6.RNAi of Tcasα6 did not result in changes in spinosad sensitivity,similar to results obtained with D.melanogaster.The lack of change in spinosad sensitivity in both D.melanogaster and T.castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species.Potential causes for the lack of change in spinosad sensitivity are discussed.

  12. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors. (United States)

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba


    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus.

  13. The diversity of GABA(A) receptor subunit distribution in the normal and Huntington's disease human brain. (United States)

    Waldvogel, H J; Faull, R L M


    GABA(A) receptors are assembled into pentameric receptor complexes from a total of 19 different subunits derived from a variety of different subunit classes (α1-6, β1-3, γ1-3, δ, ɛ, θ, and π) which surround a central chloride ion channel. GABA(A) receptor complexes are distributed heterogeneously throughout the brain and spinal cord and are activated by the extensive GABAergic inhibitory system. In this chapter, we describe the heterogeneous distribution of six of the most widely distributed subunits (α1, α2, α3, β2,3, and γ2) throughout the human basal ganglia. This review describes the studies we have carried out on the normal and Huntington's disease human basal ganglia using autoradiographic labeling and immunohistochemistry in the human basal ganglia. GABA(A) receptors are known to react to changing conditions in the brain in neurological disorders, especially in Huntington's disease and display a high degree of plasticity which is thought to compensate for loss of function caused by disease. In Huntington's disease, the variable loss of GABAergic medium spiny striatopallidal projection neurons is associated with a loss of GABA(A) receptor subunits in the striosome and/or the matrix compartments of the striatum. By contrast in the globus pallidus, a loss of the GABAergic striatal projection neurons results in a dramatic upregulation of subunits on the large postsynaptic pallidal neurons; this is thought to be a compensatory plastic mechanism resulting from the loss of striatal GABAergic input. Most interestingly, our studies have revealed that the subventricular zone overlying the caudate nucleus contains a variety of proliferating progenitor stem cells that possess a heterogeneity of GABA(A) receptor subunits which may play a role in human brain repair mechanisms.

  14. Synthesis and Pharmacology of Mono-, Di-, and Trialkyl-Substituted 7-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides Combined with X-ray Structure Analysis to Understand the Unexpected Structure-Activity Relationship at AMPA Receptors

    DEFF Research Database (Denmark)

    Larsen, Anja Probst; Francotte, Pierre; Frydenvang, Karla;


    Positive allosteric modulators of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA)-type ionotropic glutamate receptors are promising compounds for treatment of neurological disorders, for example, Alzheimer's disease. Here, we report synthesis and pharmacological evaluation...

  15. Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Hyunjeong Yang

    Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

  16. Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively

    DEFF Research Database (Denmark)

    Collins, C; Duff, C; Duncan, A M;


    A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor...... subunit (NMDAR1) and a proposed glutamate-binding subunit of the NMDA receptor (NMDARA1) have provided an opportunity to test the hypothesis that either of these genes might be directly involved in the causation of HD. We have mapped NMDAR1 to 9q34.3 using in situ hybridization studies and NMDARA1...... that the gene for torsion dystonia has also been localized by genetic studies to 9q34.3, the same regional map location as NMDAR1....

  17. Lessons from crystal structures of kainate receptors

    DEFF Research Database (Denmark)

    Møllerud, Stine; Frydenvang, Karla Andrea; Pickering, Darryl S;


    synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor...... structure and how they bind agonists, antagonists and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full...

  18. Function of Partially Duplicated Human α7 Nicotinic Receptor Subunit CHRFAM7A Gene (United States)

    de Lucas-Cerrillo, Ana M.; Maldifassi, M. Constanza; Arnalich, Francisco; Renart, Jaime; Atienza, Gema; Serantes, Rocío; Cruces, Jesús; Sánchez-Pacheco, Aurora; Andrés-Mateos, Eva; Montiel, Carmen


    The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response. PMID:21047781

  19. Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells. (United States)

    Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi


    GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca(2+) influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

  20. The paracaspase MALT1 cleaves the LUBAC subunit HOIL1 during antigen receptor signaling. (United States)

    Douanne, Tiphaine; Gavard, Julie; Bidère, Nicolas


    Antigen-receptor-mediated activation of lymphocytes relies on a signalosome comprising CARMA1 (also known as CARD11), BCL10 and MALT1 (the CBM complex). The CBM activates nuclear factor κB (NF-κB) transcription factors by recruiting the 'linear ubiquitin assembly complex' (LUBAC), and unleashes MALT1 paracaspase activity. Although MALT1 enzyme shapes NF-κB signaling, lymphocyte activation and contributes to lymphoma growth, the identity of its substrates continues to be elucidated. Here, we report that the LUBAC subunit HOIL1 (also known as RBCK1) is cleaved by MALT1 following antigen receptor engagement. HOIL1 is also constitutively processed in the 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), which exhibits aberrant MALT1 activity. We further show that the overexpression of MALT1-insensitive HOIL1 mitigates T-cell-receptor-mediated NF-κB activation and subsequent cytokine production in lymphocytes. Thus, our results unveil HOIL1 as a negative regulator of lymphocyte activation cleaved by MALT1. This cleavage could therefore constitute an appealing therapeutic target for modulating immune responses.

  1. The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues. (United States)

    Calver, A R; Medhurst, A D; Robbins, M J; Charles, K J; Evans, M L; Harrison, D C; Stammers, M; Hughes, S A; Hervieu, G; Couve, A; Moss, S J; Middlemiss, D N; Pangalos, M N


    GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

  2. Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type hori-zontal cells

    Institute of Scientific and Technical Information of China (English)


    Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or dihydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R- and G-cones to LHCs, respectively.

  3. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Directory of Open Access Journals (Sweden)

    Florian Wegner

    Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  4. Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits. (United States)

    Alzayady, Kamil J; Wagner, Larry E; Chandrasekhar, Rahul; Monteagudo, Alina; Godiska, Ronald; Tall, Gregory G; Joseph, Suresh K; Yule, David I


    Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

  5. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons. (United States)

    Son, Jong-Hyun; Winzer-Serhan, Ursula H


    Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.

  6. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling. (United States)

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N


    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  7. Environmental Risk Limits for AMPA

    NARCIS (Netherlands)

    Traas TP; Smit CE; SEC


    In dit rapport worden milieurisicogrenzen (MTR, VR en SRC-eco) afgeleid voor de stof aminomethyl-fosfonzuur (AMPA, de primaire metaboliet van het herbicide glyfosaat en fosfonaten, o.a ingredienten van wasmiddelen. De MTR voor AMPA is 79,7 ug/L. MTR's voor bodem en sediment konden niet worden

  8. Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively



    The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony- stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice...

  9. Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis

    NARCIS (Netherlands)

    Varju, P; Schlett, K; Eisel, U; Madarasz, E


    NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits.

  10. MAP1B binds to the NMDA receptor subunit NR3A and affects NR3A protein concentrations

    DEFF Research Database (Denmark)

    Eriksson, Maria; Samuelsson, Helena; Björklund, Stefan;


    Incorporation of the N-methyl-d-aspartate receptor (NMDAR) subunit NR3A into functional NMDARs results in reduced channel conductance and Ca(2+) permeability. To further investigate the function of NR3A, we have set out to characterize its intracellular binding partners. Here, we report a novel p...

  11. Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

    NARCIS (Netherlands)

    De Souza Silva, M. A.; Dolga, Amalia; Pieri, I.; Marchetti, L.; Eisel, U. L. M.; Huston, J. P.; Dere, E.


    It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-D-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the n

  12. Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lee, R.T.; Lee, Y.C.


    The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which (43 kilodalton (kDa)) exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an /sup 125/I-labeled high-affinity reagent the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. The authors postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work.

  13. Ionotropic glutamate receptor expression in preganglionic neurons of the rat inferior salivatory nucleus. (United States)

    Kim, M; Chiego, D J; Bradley, R M


    Glutamate receptor (GluR) subunit composition of inferior salivatory nucleus (ISN) neurons was studied by immunohistochemical staining of retrogradely labeled neurons. Preganglionic ISN neurons innervating the von Ebner or parotid salivary glands were labeled by application of a fluorescent tracer to the lingual-tonsilar branch of the glossopharyngeal nerve or the otic ganglion respectively. We used polyclonal antibodies to glutamate receptor subunits NR1, NR2A, NR2B, (NMDA receptor subunits) GluR1, GluR2, GluR3, GluR4 (AMPA receptor subunits), and GluR5-7, KA2 (kainate receptor subunits) to determine their expression in ISN neurons. The distribution of the NMDA, AMPA and kainate receptor subunits in retrogradely labeled ISN neurons innervating the von Ebner and parotid glands was qualitatively similar. The percentage of retrogradley labeled ISN neurons innervating the parotid gland expressing the GluR subunits was always greater than those innervating the von Ebner gland. For both von Ebner and parotid ISN neurons, NR2A subunit staining had the highest expression and the lowest expression of GluR subunit staining was NR2B for von Ebner ISN neurons and GluR1 for parotid ISN neurons. The percentage of NR2B and GluR4 expressing ISN neurons was significantly different between the two glands. The percentage of ISN neurons that expressed GluR receptor subunits ranged widely indicating that the distribution of GluR subunit expression differs amongst the ISN neurons. While ISN preganglionic neurons express all the GluR subunits, differences in the percentage of ISN neurons expression between neurons innervating the von Ebner and parotid glands may relate to the different functional roles of these glands.

  14. Putative nicotinic acetylcholine receptor subunits express differentially through the life cycle of codling moth, Cydia pomonella (Lepidoptera: Tortricidae). (United States)

    Martin, Jessica A; Garczynski, Stephen F


    Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Orchardists in Washington State are concerned about the possibility of codling moth field populations developing resistance to these two insecticides. In an effort to help mitigate this issue, we initiated a project to identify and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; (i) identify transcripts from a codling moth head transcriptome that encode for nAChR subunits, and (ii) determine nAChR subunit expression profiles in various life stages of codling moth. From a codling moth head transcriptome, 24 transcripts encoding for 12 putative nAChR subunit classes were identified and verified by PCR amplification, cloning, and sequence determination. Characterization of the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing features of the cys-loop ligand-gated ion channel superfamily with 9 α-type subunits and 3 β-type subunits identified. Phylogenetic analysis comparing these protein sequences to those of other insect nAChR subunits supports the identification of these proteins as nAChR subunits. Stage expression studies determined that there is clear differential expression of many of these subunits throughout the codling moth life cycle. The information from this study will be used in the future to monitor for potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations.

  15. 钩藤碱对甲基苯丙胺条件性位置偏爱大鼠AMPA受体蛋白改变的影响%The effect of rhynchophylline on AMPA receptors expression in methamphetamine dependent rats

    Institute of Scientific and Technical Information of China (English)

    林晓亮; 汤伟; 陈文倩; 翁建霖; 莫志贤


    Objective To study changes of AMPA receptors expression in nucleus accumbens and hypothalamus of methamphetamine dependent rats,and the therapeutical effect of rhynchophylline.Methods SPF male rata were randomly divided into normal control group,model group of methamphetamine,low dose of rhynchophylline group and high dose of rhynchophylline group(n=8 in each group).Experiment of conditioned place preference(CPP)was used to build the model of methamphetamine dependent rata.Western blotting was used to examine the changes of GluR2/3 subunits expression.The time of staying in drug-paired compartment of rats was used independent-samples t test to gather statistics,and the photodensity of proteinum strap was used One-Way ANOVA to gather statistics.Results Compare with rats in normal control group(the time of staying in drug-paired compartment of rats was(383.00±38.20)s),the rats produced CPP after treated with methamphetamine(the time of staying in drug-paired compartment of rats was(536.20±57.49)s),and low(30mg/kg) and high (60 ms/kg)dose of rhynchophylline(the time of staying in drug-paired compartment of rats were(299.80±15.96)s and(189.40±59.02)s)both could eliminate CPP effect.Compare with rats in normal control group (the ratio of value of average gray scale were(0.54±0.04)INT·mm~2 and (0.70±0.04)INT·mm~2),GluR2/3 subunits expression in nucleus aecumbens increased significantly in model group(the ratio of value of average gray seale was(0.89±0.03)INT·mm~2)and low dose of rhynchophylline group(the ratio of value of average gray seale was (0.93±0.03)INT·mm~2,P0.05).Conclusion GluR2/3 subunits expression of methamphetamine-induced CPP rats increased in nucleus accumbens but decreased in hypothalamus.High dose of rhynchophylline can reverse such changes and rebound the expression to normal level.%目的 观察甲基苯丙胺成瘾大鼠伏隔核及下丘脑中AMPA受体表达的改变及钩藤碱对其的干预作用.方法 SPF级雄性SD大鼠分为空

  16. Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit. (United States)

    Hannemann, J; Hara, T; Kawai, M; Miyajima, A; Ostertag, W; Stocking, C


    An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

  17. Spinal D1-like dopamine receptors modulate NMDA receptor-induced hyperexcitability and NR1 subunit phosphorylation at serine 889. (United States)

    Aira, Zigor; Barrenetxea, Teresa; Buesa, Itsaso; Martínez, Endika; Azkue, Jon Jatsu


    Activation of the N-methyl-d-aspartate receptor (NMDAR) in dorsal horn neurons is recognized as a fundamental mechanism of central sensitization and pathologic pain. This study assessed the influence of dopaminergic, D1-like receptor-mediated input to the spinal dorsal horn on NMDAR function. Spinal superfusion with selective NMDAR agonist cis-ACPD significantly increased C-fiber-evoked field potentials in rats subjected to spinal nerve ligation (SNL), but not in sham-operated rats. Simultaneous application of D1LR antagonist SCH 23390 dramatically reduced hyperexcitability induced by cis-ACPD. Furthermore, cis-ACPD-induced hyperexcitability seen in nerve-ligated rats could be mimicked in unin-jured rats during stimulation of D1LRs by agonist SKF 38393 at subthreshold concentration. Phosphorylation of NMDAR subunit NR1 at serine 889 at postsynaptic sites was found to be increased in dorsal horn neurons 90 min after SNL, as assessed by increased co-localization with postsynaptic marker PSD-95. Increased NR1 phosphorylation was attenuated in the presence of SCH 23390 in the spinal superfusate. The present results support that D1LRs regulate most basic determinants of NMDAR function in dorsal horn neurons, suggesting a potential mechanism whereby dopaminergic input to the dorsal horn can modulate central sensitization and pathologic pain.

  18. Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures. (United States)

    Zheng, T M; Caruncho, H J; Zhu, W J; Vicini, S; Ikonomovic, S; Grayson, D R; Costa, E


    Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

  19. PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-d-aspartate receptor subunit NR2A



    Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves N-methyl-d-aspartate (NMDA) receptor function. In this study, we examined how Fyn participates in synaptic plasticity by analyzing the physical and functional interaction between Fyn and NMDA receptors. Results showed that tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, was reduced in fyn-mutant mice. NR2A was tyrosine-phosphorylated in 293T cells when coexpre...

  20. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis. (United States)

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K


    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  1. Paradoxical proepileptic response to NMDA receptor blockade linked to cortical interneuron defect in stargazer mice

    Directory of Open Access Journals (Sweden)

    Atul eMaheshwari


    Full Text Available Paradoxical seizure exacerbation by antiepileptic medication is a well-known clinical phenomenon in epilepsy, but the cellular mechanisms remain unclear. One possibility is enhanced network disinhibition by unintended suppression of inhibitory interneurons. We investigated this hypothesis in the stargazer mouse model of absence epilepsy, which bears a mutation in stargazin, an AMPA receptor trafficking protein. If AMPA signaling onto inhibitory GABAergic neurons is impaired, their activation by glutamate depends critically upon NMDA receptors. Indeed, we find that stargazer seizures are exacerbated by NMDA receptor blockade with CPP and MK-801, whereas other genetic absence epilepsy models are sensitive to these antagonists. To determine how an AMPA receptor trafficking defect could lead to paradoxical network activation, we analyzed stargazin and AMPA receptor localization and found that stargazin is detected exclusively in parvalbumin-positive (PV+ fast-spiking interneurons in somatosensory cortex, where it is co-expressed with the AMPA receptor subunit GluA4. PV+ cortical interneurons in stargazer show a near two-fold decrease in the dendrite:soma GluA4 expression ratio compared to wild type littermates. We explored the functional consequence of this trafficking defect on network excitability in neocortical slices. Both NMDA receptor antagonists suppressed 0 Mg2+ induced network discharges in wild type but augmented bursting in stargazer cortex. Interneurons mediate this paradoxical response, since the difference between genotypes was masked by GABA receptor blockade. Our findings provide a cellular locus for AMPA receptor-dependent signaling defects in stargazer cortex and define an interneuron-dependent mechanism for paradoxical seizure exacerbation in absence epilepsy.

  2. [Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits (United States)

    Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.


    Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

  3. Lurasidone and fluoxetine reduce novelty-induced hypophagia and NMDA receptor subunit and PSD-95 expression in mouse brain. (United States)

    Stan, Tiberiu Loredan; Sousa, Vasco Cabral; Zhang, Xiaoqun; Ono, Michiko; Svenningsson, Per


    Lurasidone, a novel second-generation antipsychotic agent, exerts antidepressant actions in patients suffering from bipolar type I disorder. Lurasidone acts as a high affinity antagonist at multiple monoamine receptors, particularly 5-HT2A, 5-HT7, D2 and α2 receptors, and as a partial agonist at 5-HT1A receptors. Accumulating evidence indicates therapeutic actions by monoaminergic antidepressants are mediated via alterations of glutamate receptor-mediated neurotransmission. Here, we used mice and investigated the effects of chronic oral administration of vehicle, lurasidone (3 or 10mg/kg) or fluoxetine (20mg/kg) in the novelty induced hypophagia test, a behavioral test sensitive to chronic antidepressant treatment. We subsequently performed biochemical analyses on NMDA receptor subunits and associated proteins. Both lurasidone and fluoxetine reduced the latency to feed in the novelty-induced hypophagia test. Western blotting experiments showed that both lurasidone and fluoxetine decreased the total levels of NR1, NR2A and NR2B subunits of NMDA receptors and PSD-95 (PostSynaptic Density-95) in hippocampus and prefrontal cortex. Taken together, these data indicate that antidepressant/anxiolytic-like effects of lurasidone, as well as fluoxetine, could involve reduced NMDA receptor-mediated signal transduction, particularly in pathways regulated by PSD-95, in hippocampus and prefrontal cortex.

  4. Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption (United States)

    Ślimak, Marta A.; Ables, Jessica L.; Frahm, Silke; Antolin-Fontes, Beatriz; Santos-Torres, Julio; Moretti, Milena; Gotti, Cecilia; Ibañez-Tallon, Inés


    The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and β4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in α3β4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that β4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of β4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that β4A90I and β4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant β4D447Y, significantly increased nicotine-evoked current amplitudes, while β4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express β4 or β4 variants in the MHb. Immunoprecipitation studies confirmed that β4 lentiviral-mediated expression leads to specific upregulation of α3β4 but not β2 nAChRs in the Mhb. Mice injected with the β4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the β4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the β4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular β4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in

  5. Expression of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR2/3 receptors in the developing rat pineal gland. (United States)

    Kaur, C; Sivakumar, V; Ling, E A


    The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate (GluR2/3) receptors and N-methyl-D-aspartate receptor subtype 1 (NMDAR1) was carried out by immunohistochemistry, double immunofluorescence and real-time RT-PCR analysis in the pineal glands of 1-day to 6-wk-old rats in the present study. GluR2/3 immunopositive cells were distributed throughout the pineal gland and showed branching processes in all age groups. The NMDAR1 immunoreactivity, however, was observed in fewer branched cells. A constitutive mRNA expression of NMDAR1, GluR2 and GluR3 was detected in the pineal glands of various ages and showed no significant difference between the age groups studied. Immunohistochemical and double immunofluorescence results showed that the GluR2/3 were mainly expressed and co-localized with OX-42-positive microglia/macrophages and the glial fibrillary acidic protein (GFAP)-positive astrocytes. Co-localization of NMDAR1 with OX-42- and GFAP-positive cells was much less. The expression of these receptors on the glial cells suggests that they may be involved in the development and growth of the pineal gland in the early postnatal period (1 day to 3 wk) and subsequently in the regulation of melatonin synthesis.

  6. AMPA experimental communications systems (United States)

    Beckerman, D.; Fass, S.; Keon, T.; Sielman, P.


    The program was conducted to demonstrate the satellite communication advantages of Adaptive Phased Array Technology. A laboratory based experiment was designed and implemented to demonstrate a low earth orbit satellite communications system. Using a 32 element, L-band phased array augmented with 4 sets of weights (2 for reception and 2 for transmission) a high speed digital processing system and operating against multiple user terminals and interferers, the AMPA system demonstrated: communications with austere user terminals, frequency reuse, communications in the face of interference, and geolocation. The program and experiment objectives are described, the system hardware and software/firmware are defined, and the test performed and the resultant test data are presented.

  7. Genetic ablation of NMDA receptor subunit NR3B in mouse reveals motoneuronal and nonmotoneuronal phenotypes. (United States)

    Niemann, Stephan; Kanki, Hiroaki; Fukui, Yasuyuki; Takao, Keizo; Fukaya, Masahiro; Hynynen, Meri N; Churchill, Michael J; Shefner, Jeremy M; Bronson, Roderick T; Brown, Robert H; Watanabe, Masahiko; Miyakawa, Tsuyoshi; Itohara, Shigeyoshi; Hayashi, Yasunori


    NR3B is a modulatory subunit of the NMDA receptor, abundantly expressed in both cranial and spinal somatic motoneurons and at lower levels in other regions of the brain as well. Recently, we found the human NR3B gene (GRIN3B) to be highly genetically heterogeneous, and that approximately 10% of the normal European-American population lacks NR3B due to homozygous occurrence of a null allele in the gene. Therefore, it is especially important to understand the phenotypic consequences of the genetic loss of NR3B in both humans and animal models. We here provide results of behavioral analysis of mice genetically lacking NR3B, which is an ideal animal model due to homogeneity in genetic and environmental background. The NR3B(-/-) mice are viable and fertile. Consistent with the expression of NR3B in somatic motoneurons, the NR3B(-/-) mice showed a moderate but significant impairment in motor learning or coordination, and decreased activity in their home cages. Remarkably, the NR3B(-/-) mice showed a highly increased social interaction with their familiar cage mates in their home cage but moderately increased anxiety-like behaviour and decreased social interaction in a novel environment, consistent with the inhibitory role of NR3B on the functions of NMDA receptors. This work is the first reporting of the functional significance of NR3B in vivo and may give insight into the contribution of genetic variability of NR3B in the phenotypic heterogeneity among human population.

  8. Nicotinic Acetylcholine Receptor α4 Subunit Gene Variation Associated with Attention Deficit Hyperactivity Disorder

    Institute of Scientific and Technical Information of China (English)

    HUANG Xuezhu; XU Yong; LI Qianqian; LIU Pozi; YANG Yuan; ZHANG Fuquan; GUO Tianyou; YANG Chuang; GUO Lanting


    Previous pharmacological, human genetics, and animal models have implicated the nicotinic ace-tylcholine receptor a4 subunit (CHRNA4) gene in the pathogenesis of attention deficit/hyperactivity disorder (ADHD). The objective of this study is to examine the genetic association between single nucleotide poly-morphisms in the CHRNA4 gene (rs2273502, rs1044396, rs1044397, and rs3827020 loci) and ADHD. Both case-control and family-based designs are used. Children aged 6 to 16 years were interviewed and as-sessed with the children behavior checklist and the revised conner' parent rating scale to identify probands. No significant differences in the frequency distribution of genotypes or alleles were found between the case and control groups. However, further haplotype analyses showed the CCGG haplotype on dsk for ADHD in 164 case-control samples and the standard transmission disequilibrium test analyses suggest that the allele C of rs2273502 was over-transferred in 98 ADHD parent-offspring tdos. These findings suggest that the CHRNA4 gene may play a role in the pathogenesis of ADHD.

  9. Ca2+-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors and dopamine D1 receptors regulate GluA1 trafficking in striatal neurons. (United States)

    Tukey, David S; Ziff, Edward B


    Regulation of striatal medium spiny neuron synapses underlies forms of motivated behavior and pathological drug seeking. A primary mechanism for increasing synaptic strength is the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynapse, a process mediated by GluA1 AMPAR subunit phosphorylation. We have examined the role of converging glutamate and dopamine inputs in regulating biochemical cascades upstream of GluA1 phosphorylation. We focused on the role of Ca(2+)-permeable AMPARs (CPARs), which lack the GluA2 AMPAR subunit. Under conditions that prevented depolarization, stimulation of CPARs activated neuronal nitric oxide synthase and production of cGMP. CPAR-dependent cGMP production was sufficient to induce synaptic insertion of GluA1, detected by confocal microscopy, through a mechanism dependent on GluA1 Ser-845 phosphorylation. Dopamine D1 receptors, in contrast, stimulate GluA1 extra synaptic insertion. Simultaneous activation of dopamine D1 receptors and CPARs induced additive increases in GluA1 membrane insertion, but only CPAR stimulation augmented CPAR-dependent GluA1 synaptic insertion. This incorporation into the synapse proceeded through a sequential two-step mechanism; that is, cGMP-dependent protein kinase II facilitated membrane insertion and/or retention, and protein kinase C activity was necessary for synaptic insertion. These data suggest a feed-forward mechanism for synaptic priming whereby an initial stimulus acting independently of voltage-gated conductance increases striatal neuron excitability, facilitating greater neuronal excitation by a subsequent stimulus.

  10. Synthesis and biological evaluation of analogues of 7-chloro-4,5-dihydro-4- oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylic acid (TQX-173) as novel selective AMPA receptor antagonists. (United States)

    Catarzi, Daniela; Colotta, Vittoria; Varano, Flavia; Calabri, Francesca Romana; Filacchioni, Guido; Galli, Alessandro; Costagli, Chiara; Carlà, Vincenzo


    In recent papers (Catarzi, D.; et al. J. Med. Chem. 2000, 43, 3824-3826; 2001, 44, 3157-3165) we reported chemical and biological studies on 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylates (TQXs) bearing different nitrogen-containing heterocycles at position-8. In particular, from these studies it emerged that both the 7-chloro-4,5-dihydro-4-oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a] quinoxaline-2-carboxylic acid TQX-173 (compound B) and its corresponding ethyl ester (compound A) were the most active and selective compounds of this series. In pursuing our investigation on the structure-activity relationships of these TQX derivatives, different electron-withdrawing groups (CF(3), NO(2)) were introduced at position 7 on the TQX ring system, replacing the 7-chloro substituent of B and of other selected 8-heteroaryltriazoloquinoxaline-2-carboxylates previously described. All the newly synthesized compounds were biologically evaluated for their binding at the Gly/NMDA, AMPA, and KA high-affinity receptors. Gly/NMDA binding assays were performed to assess the selectivity of the reported compounds toward the AMPA receptor. Compounds endowed with micromolar binding affinity for the KA high-affinity binding site were also evaluated for their binding at the KA low-affinity receptor. Some selected compounds were also tested for their functional antagonist activity at the AMPA and NMDA receptor-ion channel complex. The results obtained in this study have pointed out that 4,5-dihydro-7-nitro-4-oxo-8-(3-carboxypyrrol-1-yl)-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylic acid (9b) and its corresponding ethyl ester (9a) are the most potent and selective AMPA receptor antagonists reported to date among the TQX series.

  11. Increased GABA(A receptor ε-subunit expression on ventral respiratory column neurons protects breathing during pregnancy.

    Directory of Open Access Journals (Sweden)

    Keith B Hengen

    Full Text Available GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABA(ARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABA(AR expression on brainstem neurons of the ventral respiratory column (VRC. In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABA(AR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABA(AR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABA(AR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life despite increased neurosteroid levels during pregnancy.

  12. Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist

    DEFF Research Database (Denmark)

    Elster, L; Hansen, Gert Helge; Belhage, B;


    or absence of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP). THIP (150 microM) induced a 2-fold increase in the number of alpha 1 and beta 2/3 subunits in both cell bodies and processes in 4-day-old cultures. Extending the culture period to 8 days led to a polarization...... composition. Interestingly, receptor subunit clusters, consisting of alpha 1 alone, were more frequently observed than composite (alpha 1; beta 2/3) clusters. This substantiates the view that receptors not having alpha 1 and beta 2/3 subunits in the same complex may exist....

  13. Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor. (United States)

    McQuade, R; Thomas, C G; Nayfeh, S N


    Highly purified alpha- and beta-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with Kd and Bmax values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with Mr of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH alpha-beta dimer and not to one subunit in the case of the 68,000 complex and to the TSH alpha-beta dimer in the case of the 80,000 complex, as had been hypothesized previously.

  14. Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.


    We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

  15. Three-dimensional structure of the ligand-binding core of GluR2 in complex with the agonist (S)-ATPA: implications for receptor subunit selectivity. (United States)

    Lunn, Marie-Louise; Hogner, Anders; Stensbøl, Tine B; Gouaux, Eric; Egebjerg, Jan; Kastrup, Jette S


    Two X-ray structures of the GluR2 ligand-binding core in complex with (S)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid ((S)-ATPA) have been determined with and without Zn(2+) ions. (S)-ATPA induces a domain closure of ca. 21 degrees compared to the apo form. The tert-butyl moiety of (S)-ATPA is buried in a partially hydrophobic pocket and forces the ligand into the glutamate-like binding mode. The structures provide new insight into the molecular basis of agonist selectivity between AMPA and kainate receptors.

  16. Parvalbumin immunoreactivity and expression of GABAA receptor subunits in the thalamus after experimental TBI. (United States)

    Huusko, N; Pitkänen, A


    Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy in humans, resulting in an ictogenic region that is often located in the cerebral cortex. The thalamus provides heavy projections to the cortex and the activity of thalamocortical pathways is controlled by GABAergic afferents from the reticular nucleus of the thalamus (RT). As rats with TBI induced by lateral fluid-percussion injury (FPI) undergo epileptogenesis, we hypothesized that damage to the parvalbumin (PARV)-immunoreactive (ir) neurons in the RT is associated with seizure susceptibility after lateral FPI. To address this hypothesis, adult Sprague-Dawley rats (n=13) were injured with lateral FPI. At 6months post-TBI, each animal underwent a pentylenetetrazol (PTZ) seizure susceptibility test and 2weeks of continuous video-electroencephalography (EEG) monitoring for detection of the occurrence of spontaneous seizures. Thereafter, the brain was processed for PARV immunohistochemistry. We (a) estimated the total number of PARV-ir neurons in the RT using unbiased stereology, (b) measured the volume of the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei of the thalamus, which receive PARV-ir inputs from the RT and project to the perilesional cortex, (c) quantified the density of PARV-ir terminals in the VPM-VPL, and (d) studied the expression of GABAA receptor subunits in a separate group of rats using laser-dissection of the thalamus followed by Real-Time polymerase chain reaction (RT-PCR) array studies. At 6months post-TBI, only 64% of PARV-ir neurons were remaining in the RT ipsilaterally (p0.05). Also, the volume of the VPM-VPL was only 51% of that in controls ipsilaterally (p<0.001) and 91% contralaterally (p<0.05). The density of PARV-ir axonal labeling was remarkably increased in the lateral aspects of the VPM and VPL (both p<0.001). Expression of the ε- and θ-subunits of the GABAA receptor was down-regulated (0.152, p<0.01 and 0.302, p<0.05, respectively), which could relate

  17. Association of nicotinic acetylcholine receptor subunit alpha-4 polymorphisms with smoking behaviors in Chinese male smokers

    Institute of Scientific and Technical Information of China (English)

    CHU Cheng-jing; YANG Yan-chun; WEI Jin-xue; ZHANG Lan


    Background It has been reported that the nicotinic acetylcholine receptor subunit a4 gene (CHRNA4) might be associated with smoking behaviors in the previous studies. Up to now, there are few reports on the relationship between CHRNA4 and smoking initiation. In this study, we tried to explore the role of two polymorphisms in CHRNA4 (rs 1044396 and rs 1044397) in smoking initiation and nicotine dependence in Chinese male smokers.Methods Nine hundred and sixty-six Chinese male lifetime nonsmokers and smokers were assessed by the Fagerstr(o)m test for nicotine dependence (FTND), smoking quantity (SQ) and the heaviness of smoking index (HSI). All subjects were divided into four groups based on their tobacco use history and the FTND scores. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find two polymorphisms of CHRNA4 in these subjects.Results The x2 test showed that rs1044396 was significantly associated with smoking initiation (x2=4.65, P=0.031),while both rs1044396 and rs1044397 were significantly associated with nicotine dependence (x2=5.42, P=0.020; x2=758,P=0.005). Furthermore, the T-G (3.9%) haplotype of rs1044396-rs1044397 showed significant association with smoking initiation (x2=6.30, P=0.012) and the C-G haplotype (58.9%) remained positive association with nicotine dependence (x2=8.64, P=0.003) after Bonferroni correction. The C-G haplotype also significantly increased the HSI (P=0.002) and FTND scores (P=0.001) after Bonferroni correction.Conclusion These findings suggest that CHRNA4 may be associated with smoking initiation and the C-G haplotype of rs1044396-rs1044397 might increase the vulnerability to nicotine dependence in Chinese male smokers.

  18. Group I mGluR activation reverses cocaine-induced accumulation of calcium-permeable AMPA receptors in nucleus accumbens synapses via a protein kinase C-dependent mechanism. (United States)

    McCutcheon, James E; Loweth, Jessica A; Ford, Kerstin A; Marinelli, Michela; Wolf, Marina E; Tseng, Kuei Y


    Following prolonged withdrawal from extended access cocaine self-administration in adult rats, high conductance Ca2+ -ermeable AMPA receptors (CP-AMPARs) accumulate in nucleus accumbens (NAc) synapses and mediate the expression of "incubated" cue-induced cocaine craving. Using patch-clamp recordings from NAc slices prepared after extended access cocaine self-administration and >45 d of withdrawal, we found that group I metabotropic glutamate receptor (mGluR) stimulation using 3,5-dihydroxyphenylglycine (DHPG; 50 μm) rapidly eliminates the postsynaptic CP-AMPAR contribution to NAc synaptic transmission. This is accompanied by facilitation of Ca2+ -impermeable AMPAR (CI-AMPAR)-mediated transmission, suggesting that DHPG may promote an exchange between CP-AMPARs and CI-AMPARs. In saline controls, DHPG also reduced excitatory transmission but this occurred through a CB1 receptor-dependent presynaptic mechanism rather than an effect on postsynaptic AMPARs. Blockade of CB1 receptors had no significant effect on the alterations in AMPAR transmission produced by DHPG in the cocaine group. Interestingly, the effect of DHPG in the cocaine group was mediated by mGluR1 whereas its effect in the saline group was mediated by mGluR5. These results indicate that regulation of synaptic transmission in the NAc is profoundly altered after extended access cocaine self-administration and prolonged withdrawal. Furthermore, they suggest that activation of mGluR1 may represent a potential strategy for reducing cue-induced cocaine craving in abstinent cocaine addicts.

  19. Rat neuronal nicotinic acetylcholine receptors containing a7 subunit: pharmacological properties of ligand binding and function

    Institute of Scientific and Technical Information of China (English)



    Aim: To compare pharmacological properties of heterologously expressed homomeric a7 nicotinic acetylcholine receptors (a.7 nAChRs) with those of native nAChRs containing a.7 subunit (a.7* nAChRs) in rat hippocampus and cerebral cortex. Methods: We established a stably transfected HEK-293 cell line that expresses homomeric rat a7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat a.7* nAChRs in rat hippocampus and cerebral cortex. We used [125IJ-a-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the a.7 nAChRs expressed in this cell line were studied using whole-cell current recording.Results: The newly established cell line, KXa7Rl, expresses homomeric a7 nAChRs that bind [125I]-a-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kj values of native rat a.7* nAChRs from hippocampus (Kd=0.28±0.03 nmol/L) and cerebral cortex (Kd=0.33±0.05 nmol/L). Using whole-cell current recording, the homomeric a7 nAChRs expressed in the cells were activated by acetylcholine and (-)-nicotine with EC50 values of 280±19 nmol/L and 180±40 nmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of a.7 nAChRs, a-bungarotoxin (IC5o=19±2 nmol/L) and methyllycaconitine (IC50=100±10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric a.7 nAChRs and native a.7* receptors in rat brain, but it also revealed several notable differences.Conclusion: This newly established stable cell line should be very useful for studying the properties of homomeric a7 nAChRs and comparing these properties to native a.7* nAChRs.

  20. The effects of NR2 subunit-dependent NMDA receptor kinetics on synaptic transmission and CaMKII activation.

    Directory of Open Access Journals (Sweden)

    David M Santucci


    Full Text Available N-Methyl-D-aspartic acid (NMDA receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future

  1. Characterization of the subunit structure of the thyrotropin receptor in the FRTL-5 rat thyroid cell line. (United States)

    Gennick, S E; Thomas, C G; Nayfeh, S N


    incremental cross-linking of TSH alpha-beta dimer to receptor subunits; and 2) the TSH receptor may be an oligomer composed of three different subunits, 31,000, 17,000, and 63,000, of which only the 31,000 subunit binds TSH.

  2. Effect of developmental lead exposure on synaptic plasticity and N—methyl—D—aspartate receptor subunit in rat hippocampus

    Institute of Scientific and Technical Information of China (English)

    RuanDY; SuiL


    Chronic lead(Pb) exposure is known to be associated with learning and memory,and cognitive dysfunction in children.Previous studies have demonstrated that Pb exposure may impair neuronal process underlying synaptic plasticity via a direct interaction with N-methyl-D-aspartate (NMDA) receptors(NMDARs).The studies described here were carried out to investigate effect of developmental Pb exposure on long-term potentiation(LTP),long-tern depression(LTD) and NMDAs subunits in rat hippocampus.The results are listed as follows:(1)low-level Pb exposture can impair the induction and maintenance of LTP in vivo and in vitro;(2)the Pb-induced impairment of LTD magnitude was an age-related decline in area CA1 of rat hippocampus;(3)chronic Pb exposure affected two components,voltage-gated calcium channel-dependent LTD and NMDARs-dependent LTD,of LTD induction in area CA1 of rat hippocampus;(4)different effects of developmental Pb exposure on NMDA receptor NR1,NR2A,NR2B,NR2C,NR2D and NR3A subunits in area CA1,CA2,CA3 and CA4 of rat hippocampus were observed;(5)the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors enriched in area CA1,CA3 and dentate gyrus and kainite receptors enriched in area CA1 and dentate gyrus of rat hippocampus were impaired by Pb exposure.

  3. The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits α1, β2, and δ along their extrasynaptic cell membrane. (United States)

    Milenkovic, I; Vasiljevic, M; Maurer, D; Höger, H; Klausberger, T; Sieghart, W


    Neuronal circuitries in the hippocampus are involved in navigation and memory and are controlled by major networks of GABAergic interneurons. Parvalbumin (PV)-expressing interneurons in the dentate gyrus (DG) are identified as fast-spiking cells, playing a crucial role in network oscillation and synchrony. The inhibitory modulation of these interneurons is thought to be mediated mainly through GABAA receptors, the major inhibitory neurotransmitter receptors in the brain. Here we show that all PV-positive interneurons in the granular/subgranular layer (GL/SGL) of the mouse DG express high levels of the GABAA receptor δ subunit. PV-containing interneurons in the hilus and the molecular layer, however, express the δ subunit to a lower extent. Only 8% of the somatostatin-containing interneurons express the δ subunit, whereas calbindin- or calretinin-containing interneurons in the DG seem not to express the GABAA receptor δ subunit at all. Hence, these cells receive a GABAergic control different from that of PV-containing interneurons in the GL/SGL. Experiments investigating a possible co-expression of GABAA receptor α1, α2, α3, α4, α5, β1, β2, β3, or γ2 subunits with PV and δ subunits indicated that α1 and β2 subunits are co-expressed with δ subunits along the extrasynaptic membranes of PV-interneurons. These results suggest a robust tonic GABAergic control of PV-containing interneurons in the GL/SGL of the DG via δ subunit-containing receptors. Our data are important for better understanding of the neuronal circuitries in the DG and the role of specific cell types under pathological conditions.

  4. Distinct regions within the GluN2C subunit regulate the surface delivery of NMDA receptors

    Directory of Open Access Journals (Sweden)

    Katarina eLichnerova


    Full Text Available N-methyl-D-aspartate (NMDA receptors mediate fast excitatory synaptic transmission in the mammalian central nervous system. The activation of NMDA receptors plays a key role in brain development, synaptic plasticity, and memory formation, and is a major contributor to many neuropsychiatric disorders. Here, we investigated the mechanisms that underlie the trafficking of GluN1/GluN2C receptors. Using an approach combining molecular biology, microscopy, and electrophysiology in mammalian cell lines and cultured cerebellar granule cells, we found that the surface delivery of GluN2C-containing receptors is reduced compared to GluN2A- and GluN2B-containing receptors. Furthermore, we identified three distinct regions within the N-terminus, M3 transmembrane domain, and C-terminus of GluN2C subunits that are required for proper intracellular processing and surface delivery of NMDA receptors. These results shed new light on the regulation of NMDA receptor trafficking, and these findings can be exploited to develop new strategies for treating some forms of neuropsychiatric disorders.

  5. Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABAA Receptor γ2 Subunit in the Cerebellum (United States)

    Leppä, Elli; Linden, Anni-Maija; Aller, Maria I.; Wulff, Peer; Vekovischeva, Olga; Luscher, Bernhard; Lüddens, Hartmut; Wisden, William; Korpi, Esa R.


    Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABAA receptors. In recombinant receptors and genetic mouse models their actions depend on the α, β, and δ subunits of the receptor, especially on those that form extrasynaptic GABAA receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunit-containing receptors and even on αβ binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5β-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABAA receptor-mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABAA receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5β-pregnan-3α-ol-20-one. Motor-impairing effects of 5β-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABAA responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABAA receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABAA receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic αβ or αβδ receptors lead to enhanced changes in the cerebellum-generated behavior. PMID:27833556

  6. Long-term changes in brain following continuous phencyclidine administration: An autoradiographic study using flunitrazepam, ketanserin, mazindol, quinuclidinyl benzilate, piperidyl-3,4-{sup 3}H(N)-TCP, and AMPA receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Ellison, Gaylord; Keys, Alan; Noguchi, Kevin [Univ. of California Los Angeles, Dept. of Psychology, Los Angeles, CA (United States)


    Phencyclidine induces a model psychosis which can persist for prolonged periods and presents a strong drug model of schizophrenia. When given continuously for several days to rats, phencyclidine and other N-methyl-D-aspartate (NMDA) antagonists induce neural degeneration in a variety of limbic structures, including retrosplenial cortex, hippocampus, septohippocampal projections, and piriform cortex. In an attempt to further clarify the mechanisms underlying these degeneration patterns, autoradiographic studies using a variety of receptor ligands were conducted in animals 21 days after an identical dosage of the continuous phencyclidine administration employed in the previous degeneration studies. The results indicated enduring alterations in a number of receptors: these included decreased piperidyl-3,4-{sup 3}H(N)-TCP (TCP), flunitrazepam, and mazindol binding in many of the limbic regions in which degeneration has been reported previously. Quinuclidinyl benzilate and (AMPA) binding were decreased in anterior cingulate and piriform cortex, and in accumbens and striatum. Piperidyl-3,4-{sup 3}H(N)-TCP binding was decreased in most hippocampal regions. Many of these long-term alterations would not have been predicted by prior studies of the neurotoxic effects of continuous phencyclidine, and these results do not suggest a unitary source for the neurotoxicity. Whereas retrosplenial cortex, the structure which degenerates earliest, showed minimal alterations, some of the most consistent, long term alterations were in structures which evidence no immediate signs of neural degeneration, such as anterior cingulate cortex and caudate nucleus. In these structures, some of the receptor changes appeared to develop gradually (they were not present immediately after cessation of drug administration), and thus were perhaps due to changed input from regions evidencing neurotoxicity. Some of these findings, particularly in anterior cingulate, may have implications for models of

  7. Silencing gamma-aminobutyric acid A receptor alpha 1 subunit expression and outward potassium current in developing cortical neurons

    Institute of Scientific and Technical Information of China (English)

    Tao Bo; Jiang Li; Jian Li; Xingfang Li; Kaihui Xing


    We used RNA interference (RNAi) to disrupt synthesis of the cortical neuronal γ-aminobutyric acid A receptor (GABAAR) α1 in rats during development, and measured outward K+ currents during neuronal electrical activity using whole-cell patch-clamp techniques. Three pairs of small interfering RNA (siRNA) for GABAAR α1 subunit were designed using OligoEngine RNAi software. This siRNA was found to effectively inhibited GABAAR α1 mRNA expression in cortical neuronal culture in vitro, but did not significantly affect neuronal survival. Outward K+ currents were decreased, indicating that GABAAR α1 subunits in developing neurons participate in neuronal function by regulating outward K+ current.

  8. Role of hippocampal AMPA receptors in antidepressant effect of ketamine in rats%海马AMPA受体在氯胺酮对大鼠抗抑郁效应中的作用

    Institute of Scientific and Technical Information of China (English)

    杨春; 高志勤; 杨春; 周志强; 杨建军; 徐建国


    Objective To evaluate the role of hippocampal AMPA receptors in the antidepressant effect of ketamine in rats.Methods Thirty male Wistar rats aged 2 months weighing 180-220 g were randomly divided into 3 groups (n =10 each):control group (group C); ketamine group (group K) and AMPA receptor antagonist NBQX group (group N).The animals were forced to swim for 15 min on the 1st day.On the 2nd day,NBQX 10 mg/kg was injected intrapefitoneally in group N; 30 min later,normal saline was injected intraperitoneally in group C,while ketamine 10 mg/kg was injected intraperitoneally in groups K and N.The forced swimming test was performed again for 5 min at 30 min after administration and the immobility time of the rats was recorded.Then the animals were sacrificed and the hippocampus was removed for determination of the expression of phosphorylated rapamycin (p-mTOR) and phosphorylated glutamate receptor 1 (p-GluR1).Results Compared with group C,the immobility time was significantly shortened and the expression of p-mTOR and p-GluR1 up-regulated in group K,and the immobility time was significantly shortened,the expression of p-mTOR up-regulated and the expression of p-GluR1 down-regulated in group N (P < 0.05).Compared with group K,the immobility time was significantly prolonged and the expression of p-mTOR and p-GluR1 down-regulated in group N (P < 0.05 ).Conclusion AMPA receptors in hippocampus are involved in the antidepressant effect of ketamine in rats and the inhibition of mTOR and GluR1 activities may be involved in the mechanism.%目的 评价海马α-氨基-3-羟基-5-甲基-4-异恶唑基丙酸(AMPA)受体在氯胺酮对大鼠抗抑郁效应中的作用.方法 雄性Wistar大鼠30只,2月龄,体重180~220 g,采用随机数字表法,将其随机均分为3组(n=10):对照组(C组)、氯胺酮组(K组)和AMPA受体拮抗剂NBQX组(N组).行强迫游泳实验15 min建立大鼠抑郁模型.于第2天N组腹腔注射NBQX 10 mg/kg;30 min

  9. Radiosynthesis and preliminary PET evaluation of (18)F-labeled 2-(1-(3-fluorophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl)benzonitrile for imaging AMPA receptors. (United States)

    Yuan, Gengyang; Jones, Graham B; Vasdev, Neil; Liang, Steven H


    To prompt the development of (18)F-labeled positron emission tomography (PET) tracers for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, we have prepared (18)F-labeled 2-(1-(3-fluorophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl)benzonitrile ([(18)F]8). The radiosynthesis was achieved by a one-pot two-step method that utilized a spirocyclic hypervalent iodine(III) mediated radiofluorination to prepare the (18)F-labeled 1-bromo-3-fluorobenzene ([(18)F]15) intermediate with K(18)F. A subsequent copper(I) iodide mediated coupling reaction was carried out with 2-(2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl)benzonitrile (10) to [(18)F]8 in 10±2% uncorrected radiochemical yield relative to starting (18)F-fluoride with >99% radiochemical purity and 29.6±7.4Gbq/μmol specific activity at the time of injection. PET imaging studies with the title radiotracer in normal mice demonstrated good brain uptake (peak standardized uptake value (SUV)=2.3±0.1) and warrants further in vivo validation.

  10. Tumor necrosis factor receptor-associated protein 1 improves hypoxia-impaired energy production in cardiomyocytes through increasing activity of cytochrome c oxidase subunit II. (United States)

    Xiang, Fei; Ma, Si-Yuan; Zhang, Dong-Xia; Zhang, Qiong; Huang, Yue-Sheng


    Tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes against hypoxia, but the underlying mechanisms are not completely understood. In the present study, we used gain- and loss-of-function approaches to explore the effects of tumor necrosis factor receptor-associated protein 1 and cytochrome c oxidase subunit II on energy production in hypoxic cardiomyocytes. Hypoxia repressed ATP production in cultured cardiomyocytes, whereas overexpression of tumor necrosis factor receptor-associated protein 1 significantly improved ATP production. Conversely, knockdown of tumor necrosis factor receptor-associated protein 1 facilitated the hypoxia-induced decrease in ATP synthesis. Further investigation revealed that tumor necrosis factor receptor-associated protein 1 induced the expression and activity of cytochrome c oxidase subunit II, a component of cytochrome c oxidase that is important in mitochondrial respiratory chain function. Moreover, lentiviral-mediated overexpression of cytochrome c oxidase subunit II antagonized the decrease in ATP synthesis caused by knockdown of tumor necrosis factor receptor-associated protein 1, whereas knockdown of cytochrome c oxidase subunit II attenuated the increase in ATP synthesis caused by overexpression of tumor necrosis factor receptor-associated protein 1. In addition, inhibition of cytochrome c oxidase subunit II by a specific inhibitor sodium azide suppressed the ATP sy nthesis induced by overexpressed tumor necrosis factor receptor-associated protein 1. Hence, tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxia at least partly via potentiation of energy generation, and cytochrome c oxidase subunit II is one of the downstream effectors that mediates the tumor necrosis factor receptor-associated protein 1-mediated energy generation program.

  11. Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating. (United States)

    Stephan, Gabriele; Kowalski-Jahn, Maria; Zens, Christopher; Schmalzing, Günther; Illes, Peter; Hausmann, Ralf


    P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify inter-subunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gating-induced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Charge-reversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.

  12. Removal of GABA(A receptor γ2 subunits from parvalbumin neurons causes wide-ranging behavioral alterations.

    Directory of Open Access Journals (Sweden)

    Elli Leppä

    Full Text Available We investigated the behavioral significance of fast synaptic inhibition by αβγ2-type GABA(A receptors on parvalbumin (Pv cells. The GABA(A receptor γ2 subunit gene was selectively inactivated in Pv-positive neurons by Cre/loxP recombination. The resulting Pv-Δγ2 mice were relatively healthy in the first postnatal weeks; but then as Cre started to be expressed, the mice progressively developed wide-ranging phenotypic alterations including low body weight, motor deficits and tremor, decreased anxiety levels, decreased pain sensitivity and deficient prepulse inhibition of the acoustic startle reflex and impaired spatial learning. Nevertheless, the deletion was not lethal, and mice did not show increased mortality even after one year. Autoradiography with t-butylbicyclophosphoro[(35S]thionate suggested an increased amount of GABA(A receptors with only α and β subunits in central nervous system regions that contained high levels of parvalbumin neurons. Using BAC-transgenesis, we reduced some of the Pv-Δγ2 phenotype by selectively re-expressing the wild-type γ2 subunit back into some Pv cells (reticular thalamic neurons and cerebellar Pv-positive neurons. This produced less severe impairments of motor skills and spatial learning compared with Pv-Δγ2 mice, but all other deficits remained. Our results reveal the widespread significance of fast GABAergic inhibition onto Pv-positive neurons for diverse behavioral modalities, such as motor coordination, sensorimotor integration, emotional behavior and nociception.

  13. Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Thomas Sunil


    Full Text Available Abstract Background The complement system is thought to be involved in the pathogenesis of numerous neurological diseases. We previously reported that pre-treatment of murine cortico-hippocampal neuronal cultures with the complement derived anaphylatoxin C5a, protects against glutamate mediated apoptosis. Our present study with C5a receptor knock out (C5aRKO mice corroborates that the deficiency of C5a renders C5aRKO mouse more susceptible to apoptotic injury in vivo. In this study we explored potential upstream mechanisms involved in C5a mediated neuroprotection in vivo and in vitro. Methods Based on evidence suggesting that reduced expression of glutamate receptor subunit 2 (GluR2 may influence apoptosis in neurons, we studied the effect of human recombinant C5a on GluR2 expression in response to glutamate neurotoxicity. Glutamate analogs were injected into C5aRKO mice or used to treat in vitro neuronal culture and GluR2 expression were assessed in respect with cell death. Results In C5aRKO mice we found that the neurons are more susceptible to excitotoxicity resulting in apoptotic injury in the absence of the C5a receptor compared to WT control mice. Our results suggest that C5a protects against apoptotic pathways in neurons in vitro and in vivo through regulation of GluR2 receptor expression. Conclusion Complement C5a neuroprotects through regulation of GluR2 receptor subunit.

  14. Identification and functional expression of a family of nicotinic acetylcholine receptor subunits in the central nervous system of the mollusc Lymnaea stagnalis. (United States)

    van Nierop, Pim; Bertrand, Sonia; Munno, David W; Gouwenberg, Yvonne; van Minnen, Jan; Spafford, J David; Syed, Naweed I; Bertrand, Daniel; Smit, August B


    We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the alpha-type, 1 is of the beta-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The alpha-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise approximately 10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.

  15. Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels. (United States)

    Asanov, Alexander; Sampieri, Alicia; Moreno, Claudia; Pacheco, Jonathan; Salgado, Alfonso; Sherry, Ryan; Vaca, Luis


    Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

  16. Acetylcholine receptor subunit and P-glycoprotein transcription patterns in levamisole-susceptible and -resistant Haemonchus contortus (United States)

    Sarai, Ranbir S.; Kopp, Steven R.; Coleman, Glen T.; Kotze, Andrew C.


    The mechanism of resistance to the anthelmintic levamisole in parasitic nematodes is poorly understood, although there is some evidence implicating changes in expression of nicotinic acetylcholine receptor (nAChR) subunit genes. Hence, in order to define levamisole resistance mechanisms in some Australian field-derived isolates of Haemonchus contortus we examined gene expression patterns and SNPs in nAChR subunit genes, as well as expression levels for P-glycoprotein (P-gp) and receptor ancillary protein genes, in various life stages of one levamisole-sensitive and three levamisole-resistant isolates of this species. Larvae of two isolates showed high-level resistance to levamisole (resistance ratios at the IC50 > 600) while the third isolate showed a degree of heterogeneity, with a resistance factor of only 1.1-fold at the IC50 alongside the presence of a resistant subpopulation. Transcription patterns for nAChR subunit genes showed a great degree of variability across the different life stages and isolates. The most consistent observation was the down-regulation of Hco-unc-63a in adults of all resistant isolates. Transcription of this gene was also reduced in the L3 stage of the two most resistant isolates, highlighting its potential as a resistance marker in the readily accessible free-living stages. There was down regulation of all four Hco-unc-29 paralogs in adults of one resistant isolate. There were no consistent changes in expression of P-gps or ancillary protein genes across the resistant isolates. The present study has demonstrated a complex pattern of nAChR subunit gene expression in H. contortus, and has highlighted several instances where reduced expression of subunit genes (Hco-unc-63a, Hco-unc-29) may be associated with the observed levamisole resistance. The data also suggests that it will be difficult to detect resistance using gene transcription-based methods on pooled larval samples from isolates containing only a resistant subpopulation due to

  17. Kalirin Binds the NR2B Subunit of the NMDA Receptor, Altering Its Synaptic Localization and Function

    KAUST Repository

    Kiraly, D. D.


    The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.

  18. GABA(A receptors containing the α2 subunit are critical for direction-selective inhibition in the retina.

    Directory of Open Access Journals (Sweden)

    Olivia Nicola Auferkorte

    Full Text Available Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS retinal circuits include several subtypes of ganglion cells (GCs and inhibitory interneurons, such as starburst amacrine cells (SACs. Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL of mouse and rabbit retina, GABA(A receptor subunit α2 (GABA(AR α2 aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(AR α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(AR α2 knock-out (KO mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(ARs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.

  19. Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-D-aspartate receptor subunits and D-serine. (United States)

    Savignac, Helene M; Corona, Giulia; Mills, Henrietta; Chen, Li; Spencer, Jeremy P E; Tzortzis, George; Burnet, Philip W J


    The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action. Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion. We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and

  20. In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons. (United States)

    Ferando, Isabella; Mody, Istvan


    Perisynaptic and extrasynaptic δ subunit-containing GABAA receptors (δ-GABAARs) mediate tonic conductances in many neurons. On principal cells of the neocortex and hippocampus they comprise α4 subunits, whereas they usually contain α1 on various interneurons. Specific characteristics of δ-GABAARs are their pharmacology and high plasticity. In particular δ-GABAARs are sensitive to low concentrations of neurosteroids (NS) and during times of altered NS production (stress, puberty, ovarian cycle and pregnancy) δ-GABAARs expression varies in many neurons regardless of the α subunits they contain, with direct consequences for neuronal excitability and network synchrony. For example δ-GABAARs plasticity on INs underlies modifications in hippocampal γ oscillations during pregnancy or over the ovarian cycle. Most δ-GABAAR-expressing INs in CA3 stratum pyramidale (SP) are parvalbumin (PV) + INs, whose fundamental role in γ oscillations generation and control has been extensively investigated. In this study we reduced or deleted δ-subunits in PV + INs, with the use of a PV/Cre-Gabrd/floxed genetic system. We find that in vitro CA3 γ oscillations of both PV-Gabrd(+/-)and PV-Gabrd(-/-) mice are characterized by higher frequencies than WT controls. The increased frequencies could be lowered to control levels in PV-Gabrd(+/-) by the NS allopregnanolone (3α,5α-tetrahydroprogesterone, 100 nM) but not the synthetic δ-GABAAR positive allosteric modulator 4-Chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl] benzamide (DS-2, 10 μM). This is consistent with the idea that DS-2, in contrast to ALLO, selectively targets α4/δ-GABAARs but not the α1/δ-GABAARs found on INs. Therefore, development of drugs selective for IN-specific α1/δ-GABAARs may be useful in neurological and psychiatric conditions correlated with altered PV + IN function and aberrant γ oscillations.

  1. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Machaalani, R., E-mail: [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Ghazavi, E. [Bosch Institute, The University of Sydney, NSW 2006 (Australia); School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Hinton, T. [School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Waters, K.A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Hennessy, A. [School of Medicine, University of Western Sydney, NSW 2751 (Australia); Heart Research Institute, 7 Eliza St Newtown, NSW 2042 (Australia)


    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

  2. Proteolytic fragments of laminin promote excitotoxic neurodegeneration by up-regulation of the KA1 subunit of the kainate receptor. (United States)

    Chen, Zu-Lin; Yu, Huaxu; Yu, Wei-Ming; Pawlak, Robert; Strickland, Sidney


    Degradation of the extracellular matrix (ECM) protein laminin contributes to excitotoxic cell death in the hippocampus, but the mechanism of this effect is unknown. To study this process, we disrupted laminin gamma1 (lamgamma1) expression in the hippocampus. Lamgamma1 knockout (KO) and control mice had similar basal expression of kainate (KA) receptors, but the lamgamma1 KO mice were resistant to KA-induced neuronal death. After KA injection, KA1 subunit levels increased in control mice but were unchanged in lamgamma1 KO mice. KA1 levels in tissue plasminogen activator (tPA)-KO mice were also unchanged after KA, indicating that both tPA and laminin were necessary for KA1 up-regulation after KA injection. Infusion of plasmin-digested laminin-1 into the hippocampus of lamgamma1 or tPA KO mice restored KA1 up-regulation and KA-induced neuronal degeneration. Interfering with KA1 function with a specific anti-KA1 antibody protected against KA-induced neuronal death both in vitro and in vivo. These results demonstrate a novel pathway for neurodegeneration involving proteolysis of the ECM and KA1 KA receptor subunit up-regulation.

  3. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse. (United States)

    Gocel, James; Larson, John


    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  4. NMDA antagonist, but not nNOS inhibitor, requires AMPA receptors in the ventromedial prefrontal cortex (vmPFC) to induce antidepressant-like effects

    DEFF Research Database (Denmark)

    Pereira, V. S.; Wegener, Gregers; Joca, S. R.


    Depressed individuals and stressed animals show enhanced levels of glutamate and neuronal nitric oxide synthase (nNOS) activity in limbic structures, including the vmPFC. Systemic administration of glutamatergic NMDA receptor antagonists or inhibitors of nitric oxide (NO) synthesis induces antide...

  5. Positive allosteric modulation of AMPA receptors differentially modulates the behavioural effects of citalopram in mouse models of antidepressant and anxiolytic action

    DEFF Research Database (Denmark)

    Fitzpatrick, Ciarán Martin; Larsen, Maria; Madsen, Louise


    Drugs that increase monoamine neurotransmission are effective in both anxiety and depression. The therapeutic effects of monoamine-based antidepressant drugs may involve indirect effects on neurotransmission through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR)....

  6. Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle


    Ahern, Chris A; Powers, Patricia A.; Biddlecome, Gloria H; Roethe, Laura; Vallejo, Paola; Mortenson, Lindsay; Strube, Caroline; Campbell, Kevin P.; Coronado, Roberto; Gregg, Ronald G.


    Background The multisubunit (α1S,α2-δ, β1a and γ1) skeletal muscle dihydropyridine receptor (DHPR) transduces membrane depolarization into release of Ca2+ from the sarcoplasmic reticulum (SR) and also acts as an L-type Ca2+ channel. To more fully investigate the function of the γ1 subunit in these two processes, we produced mice lacking this subunit by gene targeting. Results Mice lacking the DHPR γ1 subunit (γ1 null) survive to adulthood, are fertile and have no obvious gross phenotypic abno...

  7. Estrogen dissociates Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus. (United States)

    Cardona-Gómez, Gloria Patricia; Arango-Davila, Cesar; Gallego-Gómez, Juan Carlos; Barrera-Ocampo, Alvaro; Pimienta, Hernan; Garcia-Segura, Luis Miguel


    During cerebral ischemia, part of the damage associated with the hyperactivation of glutamate receptors results from the hyperphosphorylation of the microtubule-associated protein Tau. Previous studies have shown that estradiol treatment reduces neural damage after cerebral ischemia. Here, we show that transient occlusion of the middle cerebral artery results in the hyperphosphorylation of Tau and in a significant increase in the association of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type glutamate receptor subunits 2/3 in the hippocampus. Estradiol treatment decreased hippocampal injury, inhibited glycogen synthase kinase-3beta and decreased the hyperphosphorylation of Tau and the interaction of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. These findings suggest that ischemia produces a strong association between Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, and estradiol can exert at least part of its neuroprotective activity through inhibition of glycogen synthase kinase-3beta.

  8. Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

    Energy Technology Data Exchange (ETDEWEB)

    King, K.; Caron, M.G.; Lefkowitz, R.J. (Duke Univ. Medical Center, Durham, NC (USA)); Dohlman, H.G.; Thorner, J. (Univ. of California, Berkeley (USA))


    To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR and a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

  9. GABAB receptor subunit GB1 at the cell surface independently activates ERK1/2 through IGF-1R transactivation.

    Directory of Open Access Journals (Sweden)

    Guillaume A Baloucoune

    Full Text Available BACKGROUND: Functional GABA(B receptor is believed to require hetero-dimerization between GABA(B1 (GB1 and GABA(B2 (GB2 subunits. The GB1 extracellular domain is required for ligand binding, and the GB2 trans-membrane domain is responsible for coupling to G proteins. Atypical GABA(B receptor responses observed in GB2-deficient mice suggested that GB1 may have activity in the absence of GB2. However the underlying mechanisms remain poorly characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here, by using cells overexpressing a GB1 mutant (GB1asa with the ability to translocate to the cell surface in the absence of GB2, we show that GABA(B receptor agonists, such as GABA and Baclofen, can induce ERK1/2 phosphorylation in the absence of GB2. Furthermore, we demonstrate that GB1asa induces ERK1/2 phosphorylation through Gi/o proteins and PLC dependent IGF-1R transactivation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that GB1 may form a functional receptor at the cell surface in the absence of GB2.

  10. Mechanism and site of inhibition of AMPA receptors: substitution of one and two methyl groups at the 4-aminophenyl ring of 2,3-benzodiazepine and implications in the "E" site. (United States)

    Wang, Congzhou; Wu, Andrew; Shen, Yu-Chuan; Ettari, Roberta; Grasso, Silvana; Niu, Li


    2,3-Benzodiazepines are a well-known group of compounds for their potential antagonism against AMPA receptors. It has been previously reported that the inhibitory effect of 2,3-benzodiazepine derivatives with a 7,8-ethylenedioxy moiety can be enhanced by simply adding a chlorine atom at position 3 of the 4-aminophenyl ring. Here we report that adding a methyl group at position 3 on the 4-aminophenyl ring, termed as BDZ-11-7, can similarly enhance the inhibitory activity, as compared with the unsubstituted one or BDZ-11-2. Our kinetic studies have shown that BDZ-11-7 is a noncompetitive antagonist of GluA2Q homomeric receptors and prefers to inhibit the closed-channel state. However, adding another methyl group at position 5 on the 4-aminophenyl ring, termed as BDZ-11-6, fails to yield extra inhibition on GluA2Q receptors. Instead, BDZ-11-6 exhibits a diminished inhibition of GluA2Q. Site interaction test indicates the two compounds, BDZ-11-6 and BDZ-11-7, bind to the same site on GluA2Q, which is also the binding site for their prototype, BDZ-11-2. Based on the results from this and our earlier studies, we propose that the binding site that accommodates the 4-aminophenyl ring must contain two interactive points, with one preferring polar groups like chlorine and the other preferring nonpolar groups such as a methyl group. Either adding a chlorine or a methyl group may enhance the inhibitory activity of 2,3-benzodiazepine derivatives with a 7,8-ethylenedioxy moiety. Adding any two of the same group on positions 3 and 5 of the 4-aminophenyl ring, however, significantly reduces the interaction between these 2,3-benzodiazepines and their binding site, because one group is always repelled by one interactive point. We predict therefore that adding a chlorine atom at position 3 and a methyl group at position 5 of the 4-aminophenyl ring of 2,3-benzodiazepine derivatives with a 7,8-ethylenedioxy moiety may produce a new compound that is more potent.

  11. GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

    Directory of Open Access Journals (Sweden)

    De Blas Angel L


    Full Text Available Abstract Background Gamma-aminobutyric acid type A receptors (GABAA-Rs are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. Results Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. Conclusion We conclude that: 1 insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably

  12. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties.

    Directory of Open Access Journals (Sweden)

    Morten Skøtt Thomsen

    Full Text Available The existence of α7β2 nicotinic acetylcholine receptors (nAChRs has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.

  13. Subcellular distribution of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells within subventricular zone of adult rats

    Institute of Scientific and Technical Information of China (English)

    Zhining Li; Wenlong Lü; Hongyan Dong; Hongbin Fan; Ruiguo Dong; Tiejun Xu


    The subcellular localization of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells of the subventricular zone of adult rats was detected using electron microscopy, following immunohistochemistry and immunogold-silver double staining. Results confirmed the presence of neural stem cells in the subventricular zone, which is a key neurogenic region in the central nervous system of adult mammals. The expression of N-methyl-D-aspartic acid receptor subunit 1 was higher than that of nestin and mainly distributed in the cell membrane, cytoplasm, rough endoplasmic reticulum and Golgi complex of neural stem cells.

  14. Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction. (United States)

    Christensen, Mark H; Kohlmeier, Kristi A


    The earlier an individual initiates cigarette smoking, the higher the likelihood of development of dependency to nicotine, the addictive ingredient in cigarettes. One possible mechanism underlying this higher addiction liability is an ontogenetically differential cellular response induced by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within the laterodorsal tegmentum (LDT), a nucleus importantly involved in drug addiction associated behaviours, across two periods of ontogeny in which nicotine-mediated excitatory responses were shown to depend on age. To this end, whole-cell patch-clamp recordings in mouse brain slices from identified LDT neurons, in combination with nAChR subunit-specific receptor antagonists, were conducted. Comparison of the contribution of different nAChR subunits to acetylcholine (ACh)-induced inward currents indicated that the contributions of the β2 and/or β4 and α7 nAChR subunits alter across age. Taken together, we conclude that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny could contribute to the age-related differential excitability induced by nicotine. Differences in the subunit composition of nAChRs within the LDT would be expected to contribute to ontogenetic-dependent outflow from the LDT to target regions, which include reward-related circuitry.

  15. Delta-subunit containing GABAA-receptors mediate tonic inhibition in paracapsular cells of the mouse amygdala

    Directory of Open Access Journals (Sweden)

    Anne eMarowsky


    Full Text Available The intercalated paracapsular cells (pcs are small GABAergic interneurons that form densely populated clusters surrounding the basolateral (BLA complex of the amygdala. Their main task in the amygdala circuitry appears to be the control of information flow, as they act as an inhibitory interface between input and output nuclei. Modulation of their activity is thus thought to affect amygdala output and the generation of fear and anxiety. Recent evidence indicates that pcs express benzodiazepine (BZ-sensitive GABAA receptor (GABAAR variants containing the α2- and α3-subunit for transmission of postsynaptic currents, yet little is known about the expression of extrasynaptic GABAARs, mediating tonic inhibition and regulating neuronal excitability. Here, we show that pcs from the lateral and medial intercalated cell cluster (l- and mITC, respectively express a tonic GABAergic conductance that could be significantly increased in a concentration-dependent manner by the δ-preferring GABAAR agonist THIP (0.5-10 µM, but not by the BZ diazepam (1 µM. The neurosteroid THDOC (300 nM also increased tonic currents in pcs significantly, but only in the presence of additional GABA (5 µM. Immunohistochemical stainings revealed that both the δ-GABAAR and the α4-GABAAR subunit are expressed throughout all ITCs, while no staining for the α5-GABAAR subunit could be detected. Moreover, 1 µM THIP dampened excitability in pcs most likely by increasing shunting inhibition. In line with this, THIP significantly decreased lITC-generated inhibition in target cells residing in the BLA nucleus by 30%. Taken together these results demonstrate for the first time that pcs express a tonic inhibitory conductance mediated most likely by α4/δ-containing GABAARs. This data also suggest that δ-GABAAR targeting compounds might possibly interfere with pcs-related neuronal processes such as fear extinction.

  16. Blockade of NMDA receptors 2A subunit in the dorsal striatum impairs the learning of a complex motor skill. (United States)

    Lemay-Clermont, Julie; Robitaille, Christine; Auberson, Yves P; Bureau, Geneviève; Cyr, Michel


    Accumulating evidence proposes that the striatum, known to control voluntary movement, may also play a role in learning and memory. Striatum learning is thought to require long-lasting reorganization of striatal circuits and changes in the strength of synaptic connections during the memorization of a complex motor task. Whether the ionotropic glutamate receptor N-methyl-D-aspartate (NMDAR) contributes to the molecular mechanisms of these memory processes is still unclear. The aim of the present study was to investigate the role of striatal NMDAR and its subunit composition during the learning of the accelerating rotarod task in mice. To this end, we injected directly into the dorsal striatum of mice, via chronically implanted cannula, the NMDAR channel blocker MK-801 as well as the NR2A and NR2B subunit-selective antagonists NVP-AAM077 and Ro 25-6981, respectively, before rotarod training. There was no effect in the motor performances of mice treated with 1.0 μg/side of MK-801, 0.1 μg/side of NVP-AAM077, or 5 and 10 μg/side of Ro 25-6981. In contrast, injections of 2.5 and 5 μg/side of MK-801 or 0.5 and 1 μg/side of NVP-AAM077 impaired motor learning at Day 3 and 8. Interestingly, treatments with MK-801 and NVP-AAM077 did not alter the general motor capacities of mice as revealed by the stepping, wire suspension, and pole tests. Our study demonstrates that the NMDAR of the dorsal striatum contributes to motor learning, especially during the slow acquisition phase, and that NR2A subunits play a critical role in this process.

  17. Traumatic brain injury and the effects of diazepam, diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus

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    Meyer Rebecca C


    Full Text Available Abstract Background Excitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood. Methods We used Western blot procedures to test whether in vivo TBI in rat altered the protein expression of hippocampal GABA-A receptor subunits α1, α2, α3, α5, β3, and γ2 at 3 h, 6 h, 24 h, and 7 days post-injuy. We then used pre-injury injections of MK-801 to block calcium influx through the NMDA receptor, diltiazem to block L-type voltage-gated calcium influx, or diazepam to enhance chloride conductance, and re-examined the protein expressions of α1, α2, α3, and γ2, all of which were altered by TBI in the first study and all of which are important constituents in benzodiazepine-sensitive GABA-A receptors. Results Western blot analysis revealed no injury-induced alterations in protein expression for GABA-A receptor α2 or α5 subunits at any time point post-injury. Significant time-dependent changes in α1, α3, β3, and γ2 protein expression. The pattern of alterations to GABA-A subunits was nearly identical after diltiazem and diazepam treatment, and MK-801 normalized expression of all subunits 24 hours post-TBI. Conclusions These studies are the first to demonstrate that GABA-A receptor subunit expression is altered by TBI in vivo, and these alterations may be driven by calcium-mediated cascades in hippocampal neurons. Changes in GABA-A receptors in the hippocampus after TBI may have far-reaching consequences considering their essential importance in maintaining inhibitory balance and their extensive impact on neuronal function.

  18. Mutations in Two Genes Encoding Different Subunits of a Receptor Signaling Complex Result in an Identical Disease Phenotype (United States)

    Paloneva, Juha; Manninen, Tuula; Christman, Grant; Hovanes, Karine; Mandelin, Jami; Adolfsson, Rolf; Bianchin, Marino; Bird, Thomas; Miranda, Roxana; Salmaggi, Andrea; Tranebjærg, Lisbeth; Konttinen, Yrjö; Peltonen, Leena


    Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as “Nasu-Hakola disease,” is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype. PMID:12080485

  19. GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway

    Directory of Open Access Journals (Sweden)

    Yang Yongguang


    Full Text Available Abstract Background The proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF, a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1, RET tyrosine kinase, and neural cell adhesion molecule (NCAM have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs. However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs. Results In the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs. Conclusions GDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

  20. Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

    Directory of Open Access Journals (Sweden)

    Huitfeldt Henrik S


    Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits. Results We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level. Conclusion Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

  1. Targeted deletion of the mouse α2 nicotinic acetylcholine receptor subunit gene (Chrna2) potentiates nicotine-modulated behaviors. (United States)

    Lotfipour, Shahrdad; Byun, Janet S; Leach, Prescott; Fowler, Christie D; Murphy, Niall P; Kenny, Paul J; Gould, Thomas J; Boulter, Jim


    Baseline and nicotine-modulated behaviors were assessed in mice harboring a null mutant allele of the nicotinic acetylcholine receptor (nAChR) subunit gene α2 (Chrna2). Homozygous Chrna2(-/-) mice are viable, show expected sex and Mendelian genotype ratios, and exhibit no gross neuroanatomical abnormalities. A broad range of behavioral tests designed to assess genotype-dependent effects on anxiety (elevated plus maze and light/dark box), motor coordination (narrow bean traverse and gait), and locomotor activity revealed no significant differences between mutant mice and age-matched wild-type littermates. Furthermore, a panel of tests measuring traits, such as body position, spontaneous activity, respiration, tremors, body tone, and startle response, revealed normal responses for Chrna2-null mutant mice. However, Chrna2(-/-) mice do exhibit a mild motor or coordination phenotype (a decreased latency to fall during the accelerating rotarod test) and possess an increased sensitivity to nicotine-induced analgesia in the hotplate assay. Relative to wild-type, Chrna2(-/-) mice show potentiated nicotine self-administration and withdrawal behaviors and exhibit a sex-dependent enhancement of nicotine-facilitated cued, but not trace or contextual, fear conditioning. Overall, our results suggest that loss of the mouse nAChR α2 subunit has very limited effects on baseline behavior but does lead to the potentiation of several nicotine-modulated behaviors.

  2. Deletion of the NMDA receptor GluN2A subunit significantly decreases dendritic growth in maturing dentate granule neurons.

    Directory of Open Access Journals (Sweden)

    Timal S Kannangara

    Full Text Available It is known that NMDA receptors can modulate adult hippocampal neurogenesis, but the contribution of specific regulatory GluN2 subunits has been difficult to determine. Here we demonstrate that mice lacking GluN2A (formerly NR2A do not show altered cell proliferation or neuronal differentiation, but present significant changes in neuronal morphology in dentate granule cells. Specifically, GluN2A deletion significantly decreased total dendritic length and dendritic complexity in DG neurons located in the inner granular zone. Furthermore, the absence of GluN2A also resulted in a localized increase in spine density in the middle molecular layer, a region innervated by the medial perforant path. Interestingly, alterations in dendritic morphology and spine density were never seen in dentate granule cells located in the outer granular zone, a region that has been hypothesized to contain older, more mature, neurons. These results indicate that although the GluN2A subunit is not critical for the cell proliferation and differentiation stages of the neurogenic process, it does appear to play a role in establishing synaptic and dendritic morphology in maturing dentate granule cells localized in the inner granular zone.

  3. How Ca2+-permeable AMPA receptors, the kinase PKA, and the phosphatase PP2B are intertwined in synaptic LTP and LTD. (United States)

    Hell, Johannes W


    Both synaptic long-term potentiation (LTP) and long-term depression (LTD) are thought to be critical for memory formation. Dell'Acqua and co-workers now demonstrate that transient postsynaptic incorporation of Ca(2+)-permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is required for LTD in the exemplary hippocampal CA1 region in 2-week-old mice. Mechanistically, LTD depends on AKAP150-anchored protein kinase A (PKA) to promote the initial functional recruitment of CP-AMPARs during LTD induction and on AKAP150-anchored protein phosphatase 2B (PP2B) to trigger their subsequent removal as part of the lasting depression of synaptic transmission.

  4. Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

    Directory of Open Access Journals (Sweden)

    Sean C. Piantadosi


    Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

  5. Circadian and developmental regulation of N-methyl-d-aspartate-receptor 1 mRNA splice variants and N-methyl-d-aspartate-receptor 3 subunit expression within the rat suprachiasmatic nucleus

    DEFF Research Database (Denmark)

    Bendová, Z; Sumová, A; Mikkelsen, Jens D.


    The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase...... shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties...... of the receptor. In the brain, the distribution of NMDAR subunits depends on the region and developmental stage. Here, we report the circadian expression of the NMDAR1 subunit in the adult rat SCN and depict its splice variants that may constitute the functional receptor channel in the SCN. During ontogenesis...

  6. Attenuation of ketamine-induced impairment in verbal learning and memory in healthy volunteers by the AMPA receptor potentiator PF-04958242. (United States)

    Ranganathan, M; DeMartinis, N; Huguenel, B; Gaudreault, F; Bednar, M M; Shaffer, C L; Gupta, S; Cahill, J; Sherif, M A; Mancuso, J; Zumpano, L; D'Souza, D C


    There is a need to develop treatments for cognitive impairment associated with schizophrenia (CIAS). The significant role played by N-methyl-d-aspartate receptors (NMDARs) in both the pathophysiology of schizophrenia and in neuronal plasticity suggests that facilitation of NMDAR function might ameliorate CIAS. One strategy to correct NMDAR hypofunction is to stimulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as AMPAR and NMDAR functioning are coupled and interdependent. In rats and nonhuman primates (NHP), AMPAR potentiators reduce spatial working memory deficits caused by the nonselective NMDAR antagonist ketamine. The current study assessed whether the AMPAR potentiator PF-04958242 would attenuate ketamine-induced deficits in verbal learning and memory in humans. Healthy male subjects (n=29) participated in two randomized treatment periods of daily placebo or PF-04958242 for 5 days separated by a washout period. On day 5 of each treatment period, subjects underwent a ketamine infusion for 75 min during which the effects of PF-04958242/placebo were assessed on ketamine-induced: (1) impairments in verbal learning and recall measured by the Hopkins Verbal Learning Test; (2) impairments in working memory on a CogState battery; and (3) psychotomimetic effects measured by the Positive and Negative Syndrome Scale and Clinician-Administered Dissociative Symptoms Scale. PF-04958242 significantly reduced ketamine-induced impairments in immediate recall and the 2-Back and spatial working memory tasks (CogState Battery), without significantly attenuating ketamine-induced psychotomimetic effects. There were no pharmacokinetic interactions between PF-04958242 and ketamine. Furthermore, PF-04958242 was well tolerated. 'High-impact' AMPAR potentiators like PF-04958242 may have a role in the treatment of the cognitive symptoms, but not the positive or negative symptoms, associated with schizophrenia. The excellent concordance between the

  7. Modification of the philanthotoxin-343 polyamine moiety results in different structure-activity profiles at muscle nicotinic ACh, NMDA and AMPA receptors

    DEFF Research Database (Denmark)

    Mellor, I R; Brier, T J; Pluteanu, F;


    -isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups...... of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most...... analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor...

  8. Propofol effectively inhibits lithium-pilocarpine-induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    Institute of Scientific and Technical Information of China (English)

    Henglin Wang; Zhuoqiang Wang; Weidong Mi; Cong Zhao; Yanqin Liu; Yongan Wang; Haipeng Sun


    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory ef-fects of propofol on status epilepticus in rats were judged based on observation of behavior, elec-troencephalography and 24-hour survival rate. Propofol (12.5-100 mg/kg) improved status epilep-ticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly in-creased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with down-regulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures.

  9. Cloning and characterization of promoter and 5'-UTR of the NMDA receptor subunit ε2 : evidence for alternative splicing of 5'-non-coding exon

    NARCIS (Netherlands)

    Klein, Matthias; Pieri, Isabelle; Uhlmann, Frank; Pfizenmaier, Klaus; Eisel, Ulrich


    Using rapid amplification of cDNA ends (RACE), we have cloned the 5'-untranslated region (5'-UTR) of the N-methyl-D-aspartate receptor subunit ε2 from murine forebrain-derived mRNA. We identified two distinct types of cDNA species differing in the presence or absence of one exon sequence. Sequencing

  10. Gender and age differences in expression of GABAA receptor subunits in rat somatosensory thalamus and cortex in an absence epilepsy model. (United States)

    Li, Huifang; Huguenard, John R; Fisher, Robert S


    Absence epilepsy is more prevalent in females, but reasons for this gender asymmetry are unknown. We reported previously that perinatal treatment of Long-Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 causes a life-long increase in EEG spike-wave discharges (SWDs), correlated with decreased expression of GABA(A) receptor subunit gamma2 protein levels in thalamic reticular and ventrobasal nuclei (SS thalamus) [Li, H., Kraus, A., Wu, J., Huguenard, J.R., Fisher, R.S., 2006. Selective changes in thalamic and cortical GABA(A) receptor subunits in a model of acquired absence epilepsy in the rat. Neuropharmacology 51, 121-128]. In this study, we explored time course and gender different effects of perinatal AY9944 treatment on expression of GABA(A) receptor alpha1 and gamma2 subunits in SS thalamus and SS cortex. Perinatal AY9944 treatment-induced decreases in GABA(A) gamma2 receptor subunits in rat SS thalamus and increases in SS cortex are gender and age specific. The findings suggest a mechanism for the higher prevalence of absence epilepsy in female patients.

  11. Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

    DEFF Research Database (Denmark)

    Meibom, K L; Kallipolitis, B H; Ebright, R H


    At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer...

  12. Cloning and characterization of promoter and 5 '-UTR of the NMDA receptor subunit epsilon(2) : evidence for alternative splicing of 5 '-non-coding exon

    NARCIS (Netherlands)

    Klein, M; Pieri, [No Value; Uhlmann, F; Pfizenmaier, K; Eisel, U


    Using rapid amplification of cDNA ends (RACE), we have cloned the 5'-untranslated region (5'-UTR) of the N-methyl-D-aspartate receptor subunit epsilon(2) from murine forebrain-derived mRNA. We identified two distinct types of cDNA species differing in the presence or absence of one exon sequence. Se

  13. 17β-Estradiol Regulation of the mRNA Expression of T-type Calcium Channel subunits: Role of Estrogen Receptor α and Estrogen Receptor β (United States)

    Bosch, Martha A.; Hou, Jingwen; Fang, Yuan; Kelly, Martin J.; Rønnekleiv., Oline K.


    Low voltage-activated (T-type) calcium channels are responsible for burst firing and transmitter release in neurons and are important for exocytosis and hormone secretion in pituitary cells. T-type channels contain an α1 subunit, of which there are three subtypes, Cav3.1, 3.2 and 3.3, and each subtype has distinct kinetic characteristics. Although 17β-estradiol modulates T-type calcium channel expression and function, little is known about the molecular mechanisms involved. Presently, we used real-time PCR quantification of RNA extracted from hypothalamic nuclei and pituitary in vehicle and E2-treated C57BL/6 mice to elucidate E2-mediated regulation of Cav3.1, 3.2 and 3.3 subunits. The three subunits were expressed in both the hypothalamus and the pituitary. E2 treatment increased the mRNA expression of Cav3.1 and 3.2, but not Cav3.3, in the medial preoptic area and the arcuate nucleus. In the pituitary, Cav3.1 was increased with E2-treatment and Cav3.2 and 3.3 were decreased. In order to examine whether the classical estrogen receptors (ERs) were involved in the regulation, we used ERα- and ERβ-deficient C57BL/6 mice and explored the effects of E2 on T-type channel subtypes. Indeed, we found that the E2-induced increase in Cav3.1 in the hypothalamus was dependent on ERα, whereas the E2 effect on Cav3.2 was dependent on both ERα and ERβ. However, the E2-induced effects in the pituitary were dependent on only the expression of ERα. The robust E2-regulation of the T-type calcium channels could be an important mechanism by which E2 increases the excitability of hypothalamic neurons and modulates pituitary secretion. PMID:19003958

  14. BDNF-induced synaptic delivery of AMPAR subunits is differentially dependent on NMDA receptors and requires ERK. (United States)

    Li, Wei; Keifer, Joyce


    Previous studies using an in vitro model of eyeblink classical conditioning in turtles suggest that increased numbers of synaptic AMPARs supports the acquisition and expression of conditioned responses (CRs). Brain-derived neurotrophic factor (BDNF) and its associated receptor tyrosine kinase, TrkB, is also required for acquisition of CRs. Bath application of BDNF alone induces synaptic delivery of GluR1- and GluR4-containing AMPARs that is blocked by coapplication of the receptor tyrosine kinase inhibitor K252a. The molecular mechanisms involved in BDNF-induced AMPAR trafficking remain largely unknown. The aim of this study was to determine whether BDNF-induced synaptic AMPAR incorporation utilizes similar cellular mechanisms as AMPAR trafficking that occurs during in vitro classical conditioning. Using pharmacological blockade and confocal imaging, the results show that synaptic delivery of GluR1 subunits during conditioning or BDNF application does not require activity of NMDARs but is mediated by extracellular signal-regulated kinase (ERK). In contrast, synaptic delivery of GluR4-containing AMPARs during both conditioning and BDNF application is NMDAR- as well as ERK-dependent. These findings indicate that BDNF application mimics AMPAR trafficking observed during conditioning by activation of some of the same intracellular signaling pathways and suggest that BDNF is a key signal transduction element in postsynaptic events that mediate conditioning.

  15. Changes in Glutamate/NMDA Receptor Subunit 1 Expression in Rat Brain after Acute and Subacute Exposure to Methamphetamine

    Directory of Open Access Journals (Sweden)

    Walailuk Kerdsan


    Full Text Available Methamphetamine (METH is a psychostimulant drug of abuse that produces long-term behavioral changes including behavioral sensitization, tolerance, and dependence. METH has been reported to induce neurotoxic effects in several areas of the brain via the dopaminergic system. Changes of dopamine function can induce malfunction of the glutamatergic system. Therefore, the aim of the present study was to examine the effects of METH administration on the expression of glutamate N-methyl-D-aspartate receptor subunit 1 (NMDAR1 in frontal cortex, striatum, and hippocampal formation after acute and subacute exposure to METH by western blotting. Male Sprague-Dawley rats were injected intraperitoneally with a single dose of 8 mg/kg METH, 4 mg/kg/day METH for 14 days and saline in acute, subacute, and control groups, respectively. A significant increase in NMDAR1 immunoreactive protein was found in frontal cortex in the subacute group (P=.036 but not in the acute group (P=.580. Moreover, a significant increase in NMDAR1 was also observed in striatum in both acute (P=.025 and subacute groups (P=.023. However, no significant differences in NMDAR1 in hippocampal formation were observed in either acute or subacute group. The results suggest that an upregulation of NMDA receptor expression may be a consequence of glutamatergic dysfunction induced by METH.

  16. NMDA receptor subunit composition determines beta-amyloid-induced neurodegeneration and synaptic loss


    Tackenberg, C; Grinschgl, S; Trutzel, A; Santuccione, A C; Frey, M C; Konietzko, U; Grimm, J.; Brandt, R.; Nitsch, R M


    Aggregates of amyloid-beta (Aβ) and tau are hallmarks of Alzheimer's disease (AD) leading to neurodegeneration and synaptic loss. While increasing evidence suggests that inhibition of N-methyl--aspartate receptors (NMDARs) may mitigate certain aspects of AD neuropathology, the precise role of different NMDAR subtypes for Aβ- and tau-mediated toxicity remains to be elucidated. Using mouse organotypic hippocampal slice cultures from arcAβ transgenic mice combined with Sindbis virus-mediated ex...

  17. Functional expression of the GABAA receptor alpha2 and alpha3 subunits at synapses between intercalated medial paracapsular neurons of mouse amygdala

    Directory of Open Access Journals (Sweden)

    Raffaella eGeracitano


    Full Text Available In the amygdala, GABAergic neurons in the intercalated medial paracapsular cluster (Imp have been suggested to play a key role in fear learning and extinction. These neurons project to the central amygdaloid nucleus and to other areas within and outside the amygdala. In addition, they give rise to local collaterals that innervate other neurons in the Imp. Several drugs, including benzodiazepines, are allosteric modulators of GABA-A receptors. Benzodiazepines have both anxiolytic and sedative actions, which are mediated through GABA-A receptors containing alpha2/3 and alpha1 subunits, respectively. To establish whether alpha1 or alpha2/3 subunits are expressed at Imp cell synapses, we used paired recordings of anatomically-identified Imp neurons and high resolution immunocytochemistry in the mouse. We observed that a selective alpha3 subunit agonist, TP003 (100 nM, significantly increased the decay time constant of the unitary IPSCs. A similar effect was also induced by zolpidem (10 microM or by diazepam (1 microM. In contrast, lower doses of zolpidem (0.1-1 microM did not significantly alter the kinetics of the unitary IPSCs. Accordingly, immunocytochemical experiments established that the alpha2 and alpha3, but not the alpha1 subunits of the GABA-A receptors, were present at Imp cell synapses of the mouse amygdala. These results define, for the first time, some of the functional GABA-A receptor subunits expressed at synapses of Imp cells. The data also provide an additional rationale to prompt the search of GABA-A receptor alpha3 selective ligands as improved anxiolytic drugs.

  18. Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

    DEFF Research Database (Denmark)

    Kachel, Hamid S; Patel, Rohit N; Franzyk, Henrik;


    in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at -80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ...... was most sensitive to PhTX-12 along with α3β4 (IC50 values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage...

  19. Antidepressant-like activity of magnesium in the chronic mild stress model in rats: alterations in the NMDA receptor subunits. (United States)

    Pochwat, Bartłomiej; Szewczyk, Bernadeta; Sowa-Kucma, Magdalena; Siwek, Agata; Doboszewska, Urszula; Piekoszewski, Wojciech; Gruca, Piotr; Papp, Mariusz; Nowak, Gabriel


    Recent data suggests that the glutamatergic system is involved in the pathophysiology and treatment of major depressive disorder (MDD) and that the N-methyl-D-aspartate (NMDA) receptor is a potential target for antidepressant drugs. The magnesium ion blocks the ion channel of the NMDA receptor and prevents its excessive activation. Some preclinical and clinical evidence suggests also that magnesium may be useful in the treatment of depression. The present study investigated the effect of magnesium treatment (10, 15 and 20 mg/kg, given as magnesium hydroaspartate) in the chronic mild stress (CMS) model of depression in rats. Moreover, the effect of CMS and magnesium (with an effective dose) on the level of the proteins related to the glutamatergic system (GluN1, GluN2A, GluN2B and PSD-95) in the hippocampus, prefrontal cortex (PFC) and amygdala were examined. A significant reduction in the sucrose intake induced by CMS was increased by magnesium treatment at a dose of 15 mg/kg, beginning from the third week of administration. Magnesium did not affect this behavioural parameter in the control animals. CMS significantly increased the level of the GluN1 subunit in the amygdala (by 174%) and GluN2A in the hippocampus (by 191%), both of which were significantly attenuated by magnesium treatment. Moreover, magnesium treatment in CMS animals increased the level of GluN2B (by 116%) and PSD-95 (by 150%) in the PFC. The present results for the first time demonstrate the antidepressant-like activity of magnesium in the animal model of anhedonia (CMS), thus indicating the possible involvement of the NMDA/glutamatergic receptors in this activity.

  20. Gamma-aminobutyric acid promotes human hepatocellular carcinoma growth through overexpressed gamma-aminobutyric acid A receptor α3 subunit

    Institute of Scientific and Technical Information of China (English)


    AIM: To investigate the expression pattern of gamma-aminobutyric acid A (GABAA) receptors in hepatocellular carcinoma (HCC) and indicate the relationship among gamma-aminobutyric acid (GABA), gamrna-aminobutyric acid A receptor α3 subunit (GABRA3) and HCC.METHODS: HCC cell line Chang, HepG2, normal liver cell line L-02 and 8 samples of HCC tissues and paired non-cancerous tissues were analyzed with semiquantitative polymerase chain reaction (PCR) for the expression of GABAA receptors. HepG2 cells were treated with gamma-aminobutyric acid (GABA) at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell doubling time test, colon formation assay, cell cycle analysis and tumor planted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRA3 in HepG2. oliferating abilities of these cells treated with or without GABA were analyzed.RESULTS: We identified the overexpression of GABRA3 in HCC cells. Knockdown of endogenous GABRA3 expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We determined the in vitro and in vivo effect of GABA in the proliferation of GABRA3-positive cell lines, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRA3-expressing HepG2 cells, but not GABRA3-knockdown HepG2 cells. This means that GABA stimulates HepG2 cell growth through GABRA3. CONCLUSION: GABA and GABRA3 play important roles in HCC development and progression and can be a promising molecular target for the development of new diagnostic and therapeutic strategies for HCC.

  1. Redistribution of ionotropic glutamate receptors detected by laser microdissection of the rat dentate gyrus 48 h following LTP induction in vivo.

    Directory of Open Access Journals (Sweden)

    Jeremy T T Kennard

    Full Text Available The persistence and input specificity of long-term potentiation (LTP make it attractive as a mechanism of information storage. In its initial phase, both in vivo and in vitro studies have shown that LTP is associated with increased membrane localization of AMPA receptor subunits, but the molecular basis of LTP maintenance over the long-term is still unclear. We have previously shown that expression of AMPA and NMDA receptor subunits is elevated in whole homogenates prepared from dentate gyrus 48 h after LTP induction in vivo. In the present study, we utilized laser microdissection (LMD techniques to determine whether AMPA and NMDA receptor upregulation occurs specifically in the stimulated regions of the dentate gyrus dendritic arbor. Receptor proteins GluN1, GluA1 and GluA2, as well as postsynaptic density protein of 95 kDa and tubulin were detected by Western blot analysis in microdissected samples. Gradients of expression were observed for GluN1 and GluA2, decreasing from the inner to the outer zones of the molecular layer, and were independent of LTP. When induced at medial perforant path synapses, LTP was associated with an apparent specific redistribution of GluA1 and GluN1 to the middle molecular layer that contains these synapses. These data indicate that glutamate receptor proteins are delivered specifically to dendritic regions possessing LTP-expressing synapses, and that these changes are preserved for at least 48 h.

  2. Conserved expression of the glutamate NMDA receptor 1 subunit splice variants during the development of the Siberian hamster suprachiasmatic nucleus.

    Directory of Open Access Journals (Sweden)

    Giles E Duffield

    Full Text Available Glutamate neurotransmission and the N-methyl-D-aspartate receptor (NMDAR are central to photic signaling to the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN. NMDARs also play important roles in brain development including visual input circuits. The functional NMDAR is comprised of multiple subunits, but each requiring the NR1 subunit for normal activity. The NR1 can be alternatively spliced to produce isoforms that confer different functional properties on the NMDAR. The SCN undergoes extensive developmental changes during postnatal life, including synaptogenesis and acquisition of photic signaling. These changes are especially important in the highly photoperiodic Siberian hamster, in which development of sensitivity to photic cues within the SCN could impact early physiological programming. In this study we examined the expression of NR1 isoforms in the hamster at different developmental ages. Gene expression in the forebrain was quantified by in situ hybridization using oligonucleotide probes specific to alternatively spliced regions of the NR1 heteronuclear mRNA, including examination of anterior hypothalamus, piriform cortex, caudate-putamen, thalamus and hippocampus. Gene expression analysis within the SCN revealed the absence of the N1 cassette, the presence of the C2 cassette alone and the combined absence of C1 and C2 cassettes, indicating that the dominant splice variants are NR1-2a and NR1-4a. Whilst we observe changes at different developmental ages in levels of NR1 isoform probe hybridization in various forebrain structures, we find no significant changes within the SCN. This suggests that a switch in NR1 isoform does not underlie or is not produced by developmental changes within the hamster SCN. Consistency of the NR1 isoforms would ensure that the response of the SCN cells to photic signals remains stable throughout life, an important aspect of the function of the SCN as a responder to

  3. Conserved expression of the glutamate NMDA receptor 1 subunit splice variants during the development of the Siberian hamster suprachiasmatic nucleus. (United States)

    Duffield, Giles E; Mikkelsen, Jens D; Ebling, Francis J P


    Glutamate neurotransmission and the N-methyl-D-aspartate receptor (NMDAR) are central to photic signaling to the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). NMDARs also play important roles in brain development including visual input circuits. The functional NMDAR is comprised of multiple subunits, but each requiring the NR1 subunit for normal activity. The NR1 can be alternatively spliced to produce isoforms that confer different functional properties on the NMDAR. The SCN undergoes extensive developmental changes during postnatal life, including synaptogenesis and acquisition of photic signaling. These changes are especially important in the highly photoperiodic Siberian hamster, in which development of sensitivity to photic cues within the SCN could impact early physiological programming. In this study we examined the expression of NR1 isoforms in the hamster at different developmental ages. Gene expression in the forebrain was quantified by in situ hybridization using oligonucleotide probes specific to alternatively spliced regions of the NR1 heteronuclear mRNA, including examination of anterior hypothalamus, piriform cortex, caudate-putamen, thalamus and hippocampus. Gene expression analysis within the SCN revealed the absence of the N1 cassette, the presence of the C2 cassette alone and the combined absence of C1 and C2 cassettes, indicating that the dominant splice variants are NR1-2a and NR1-4a. Whilst we observe changes at different developmental ages in levels of NR1 isoform probe hybridization in various forebrain structures, we find no significant changes within the SCN. This suggests that a switch in NR1 isoform does not underlie or is not produced by developmental changes within the hamster SCN. Consistency of the NR1 isoforms would ensure that the response of the SCN cells to photic signals remains stable throughout life, an important aspect of the function of the SCN as a responder to environmental changes

  4. AFM imaging reveals the assembly of a P2X receptor complex containing P2X2, P2X4 and P2X6 subunits



    Seven P2X purinergic receptor subunits have been identified: P2X1-P2X7. All except P2X6 assemble as homotrimers, and six heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6 and P2X4/6) have been described. In addition, P2X4 homomers associate with P2X2 or P2X7 homomers as dimers of trimers. The various P2X receptors show individual functional properties, suggesting distinct physiological roles. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different ce...

  5. Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory. (United States)

    Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy


    Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways.

  6. Stimulation of lateral hypothalamic kainate receptors selectively elicits feeding behavior. (United States)

    Hettes, Stacey R; Heyming, Theodore W; Stanley, B Glenn


    Glutamate and its receptor agonists, NMDA, AMPA, and KA, elicit feeding when microinjected into the lateral hypothalamus (LH) of satiated rats. However, determining the relative contributions of AMPA receptors (AMPARs) and KA receptors (KARs) to LH feeding mechanisms has been difficult due to a lack of receptor selective agonists and antagonists. Furthermore, LH injection of KA produces behavioral hyperactivity, questioning a role for KARs in feeding selective stimulation. In the present study, we used the KAR agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), which selectively binds the GluR5 subunit of KARs, to stimulate feeding, presumably via KAR activation. Using ATPA, we tested whether: (1) LH injection of ATPA elicits feeding, (2) prior treatment with the non-selective AMPA/KAR antagonist, CNQX, suppresses ATPA-elicited feeding, and (3) LH injection of ATPA elicits behavioral patterns specific for feeding. We found that injection of ATPA (0.1 and 1 nmol) elicited an intense feeding response (e.g., 4.8+/-1.6 g) that was blocked by LH pretreatment with CNQX, but was unaffected by pretreatment with the AMPAR selective antagonist, GYKI 52466. Furthermore, minute-by-minute behavioral analysis revealed that LH injection of ATPA increased time spent feeding to 55% of the initial test period with little or no effects on other behaviors at any time. In contrast, LH injection of KA similarly increased feeding but also produced intense locomotor activity. These data suggest that selective activation of LH KARs containing GluR5 subunit(s) is sufficient to elicit feeding.

  7. Individual phases of contextual fear conditioning differentially modulate dorsal and ventral hippocampal GluA1-3, GluN1-containing receptor complexes and subunits. (United States)

    Sase, Sunetra; Sase, Ajinkya; Sialana, Fernando J; Gröger, Marion; Bennett, Keiryn L; Stork, Oliver; Lubec, Gert; Li, Lin


    In contextual fear conditioning (CFC), the use of pharmacological and lesion approaches has helped to understand that there are differential roles for the dorsal hippocampus (DH) and the ventral hippocampus (VH) in the acquisition, consolidation and retrieval phases. Concomitant analysis of the DH and the VH in individual phases with respect to α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors and N-methyl-D-aspartate receptor subtype N1 (GluN1)-containing complexes (RCC) and subunits has not been reported so far. Herein, CFC was performed in mice that were euthanized at different time points. DH and VH samples were taken for the determination of RCC and subunit levels using BN- and SDS-PAGE, respectively, with subsequent Western blotting. Evaluation of spine densities, morphology, and immunohistochemistry of GluA1 and GluA2 was performed. In the acquisition phase levels of GluA1-RCC and subunits in VH were increased. In the consolidation phase GluA1- and GluA2-RCC levels were increased in DH and VH, while both receptor subunit levels were increased in the VH only. In the retrieval phase GluA1-RCC, subunits thereof and GluA2-RCC were increased in DH and VH, whereas GluA2 subunits were increased in the VH only. GluN1-RCC levels were increased in acquisition and consolidation phase, while subunit levels in the acquisition phase were increased only in the DH. The immunohistochemical studies in the individual phases in subareas of hippocampus supported immunochemical changes of GluA1 and GluA2 RCC's. Dendritic spine densities and the prevalence of thin spines in the acquisition phase of VH and mushroom spines in the retrieval phase of the VH and DH were increased. The findings from the current study suggest different receptor and receptor complex patterns in the individual phases in CFC and in DH and VH. The results propose that different RCCs are formed in the individual phases and that VH and DH may be involved in CFC.

  8. Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit. (United States)

    Keum, Dongil; Baek, Christina; Kim, Dong-Il; Kweon, Hae-Jin; Suh, Byung-Chang


    G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca(2+), and phosphatidylinositol 4,5-bisphosphate (PIP2), to modulate N-type voltage-gated Ca(2+) (CaV2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which GqPCRs inhibit CaV2.2 channel current are not completely understood. Here, we report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membrane-localized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of

  9. Transformation of postingestive glucose responses after deletion of sweet taste receptor subunits or gastric bypass surgery. (United States)

    Geraedts, Maartje C P; Takahashi, Tatsuyuki; Vigues, Stephan; Markwardt, Michele L; Nkobena, Andongfac; Cockerham, Renee E; Hajnal, Andras; Dotson, Cedrick D; Rizzo, Mark A; Munger, Steven D


    The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) + type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K(+) (K(ATP)) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3(-/-) mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3(+/+), but not T1R3(-/-), ileum explants; this secretion was not mimicked by the K(ATP) channel blocker glibenclamide. T1R2(-/-) mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was K(ATP) channel-dependent and glucose-specific emerged in the large intestine of T1R3(-/-) mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.

  10. Lessons from crystal structures of kainate receptors. (United States)

    Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm


    Kainate receptors belong to the family of ionotropic glutamate receptors. These receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor structure and how they bind agonists, antagonists and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full-length structure has been determined of GluK2 by cryo electron microscopy to 7.6 Å resolution as well as 84 high-resolution crystal structures of N-terminal domains and ligand-binding domains, including agonist and antagonist bound structures, modulatory ions and mutations. However, there are still many unanswered questions and challenges in front of us. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

  11. The C. elegans nuclear receptor gene fax-1 and homeobox gene unc-42 coordinate interneuron identity by regulating the expression of glutamate receptor subunits and other neuron-specific genes. (United States)

    Wightman, Bruce; Ebert, Bryan; Carmean, Nicole; Weber, Katherine; Clever, Sheila


    The fax-1 gene of the nematode C. elegans encodes a conserved nuclear receptor that is the ortholog of the human PNR gene and functions in the specification of neuron identities. Mutations in fax-1 result in locomotion defects. FAX-1 protein accumulates in the nuclei of 18 neurons, among them the AVA, AVB, and AVE interneuron pairs that coordinate body movements. The identities of AVA and AVE interneurons are defective in fax-1 mutants; neither neuron expresses the NMDA receptor subunits nmr-1 and nmr-2. Other ionotropic glutamate receptor subunits are expressed normally in the AVA and AVE neurons. The unc-42 homeobox gene also regulates AVA and AVE identity; however, unc-42 mutants display the complementary phenotype: NMDA receptor subunit expression is normal, but some non-NMDA glutamate receptor subunits are not expressed. These observations support a combinatorial role for fax-1 and unc-42 in specifying AVA and AVE identity. However, in four other neuron types, fax-1 is regulated by unc-42, and both transcriptional regulators function in the regulation of the opt-3 gene in the AVE neurons and the flp-1 and ncs-1 genes in the AVK neurons. Therefore, while fax-1 and unc-42 act in complementary parallel pathways in some cells, they function in overlapping or linear pathways in other cellular contexts, suggesting that combinatorial relationships among transcriptional regulators are complex and cannot be generalized from one neuron type to another.

  12. Ganglioside Regulation of AMPA Receptor Trafficking


    Prendergast, Jillian; Umanah, George K. E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.; Schnaar, Ronald L.


    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside G...

  13. Stat2 binding to the interferon-alpha receptor 2 subunit is not required for interferon-alpha signaling. (United States)

    Nguyen, Vinh-Phúc; Saleh, Abu Z M; Arch, Allison E; Yan, Hai; Piazza, Flavia; Kim, John; Krolewski, John J


    The interferon-alpha (IFNalpha) receptor consists of two subunits, the IFNalpha receptor 1 (IFNaR1) and 2 (IFNaR2) chains. Following ligand binding, IFNaR1 is phosphorylated on tyrosine 466, and this site recruits Stat2 via its SH2 domain. In contrast, IFNaR2 binds Stat2 constitutively. In this study we have characterized the Stat2-IFNaR2 interaction and examined its role in IFNalpha signaling. Stat2 binds the major IFNaR2 protein but not a variant containing a shorter cytoplasmic domain. The interaction does not require a STAT SH2 domain. Both tyrosine-phosphorylated and non-phosphorylated Stat2 bind IFNaR2 in vitro; however, relatively little phosphorylated Stat2 associates with IFNaR2 in vivo. In vitro binding assays defined IFNaR2 residues 418-444 as the minimal interaction domain and site-specific mutation of conserved acidic residues within this domain disrupted in vitro and in vivo binding. An IFNaR2 construct carrying these mutations was either (i) overexpressed in 293T cells or (ii) used to complement IFNaR2-deficient U5A cells. Unexpectedly, the activity of an IFNalpha-dependent reporter gene was not reduced but, instead, was enhanced up to 2-fold. This suggests that this particular IFNaR2-Stat2 interaction is not required for IFNalpha signaling, but might act to negatively inhibit signaling. Finally, a doubly truncated recombinant fragment of Stat2, spanning residues 136-702, associated with IFNaR2 in vitro, indicating that the interaction with IFNaR2 is direct and occurs in a central region of Stat2 marked by a hydrophobic core.

  14. Using concatenated subunits to investigate the functional consequences of heterotetrameric inositol 1,4,5-trisphosphate receptors. (United States)

    Chandrasekhar, Rahul; Alzayady, Kamil J; Yule, David I


    Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of ubiquitous, ER localized, tetrameric Ca2+ release channels. There are three subtypes of the IP3Rs (R1, R2, R3), encoded by three distinct genes, that share ∼60-70% sequence identity. The diversity of Ca2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors, most significantly InsP3, Ca2+ and ATP. However, largely unexplored is the notion of additional signal diversity arising from the assembly of both homo and heterotetrameric InsP3Rs. In the present article, we review the biochemical and functional evidence supporting the existence of homo and heterotetrameric populations of InsP3Rs. In addition, we consider a strategy that utilizes genetically concatenated InsP3Rs to study the functional characteristics of heterotetramers with unequivocally defined composition. This approach reveals that the overall properties of IP3R are not necessarily simply a blend of the constituent monomers but that specific subtypes appear to dominate the overall characteristics of the tetramer. It is envisioned that the ability to generate tetramers with defined wild type and mutant subunits will be useful in probing fundamental questions relating to IP3R structure and function.

  15. The contribution of delta subunit-containing GABAA receptors to phasic and tonic conductance changes in cerebellum, thalamus and neocortex.

    Directory of Open Access Journals (Sweden)

    Stephen G Brickley


    Full Text Available We have made use of the delta subunit-selective allosteric modulator DS2 (4-chloro-N-[2-(2-thienylimidazo[1,2-a]pyridine-3-yl benzamide to assay the contribution of delta-GABAARs to tonic and phasic conductance changes in the cerebellum, thalamus and neocortex. In cerebellar granule cells, an enhancement of the tonic conductance was observed for DS2 and the orthosteric agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. As expected, DS2 did not alter the properties of GABAA receptor-mediated inhibitory postsynaptic synaptic currents (IPSCs supporting a purely extrasynaptic role for delta-GABAARs in cerebellar granule cells. DS2 also enhanced the tonic conductance recorded from thalamic relay neurons of the visual thalamus with no alteration in IPSC properties. However, in addition to enhancing the tonic conductance DS2 also slowed the decay of IPSCs recorded from layer II/III neocortical neurons. A slowing of the IPSC decay also occurred in the presence of the voltage-gated sodium channel blocker TTX. Moreover, under conditions of reduced GABA release the ability of DS2 to enhance the tonic conductance was attenuated. These results indicate that delta-GABAARs can be activated following vesicular GABA release onto neocortical neurons and that the actions of DS2 on the tonic conductance may be influenced by the ambient GABA levels present in particular brain regions.

  16. Antibody against recombinant heat labile enterotoxin B subunit (rLTB could block LT binding to ganglioside M1 receptor

    Directory of Open Access Journals (Sweden)

    SM Moazzeni


    Full Text Available Objectives: Enterotoxigenic Escherichia coli (ETEC is one of the most common agents of diarrhea among other bacterial agents. Designing and producing vaccine against these bacteria is one of the major purposes of World Health Organization (WHO. Due to presence of diverse clones of ETEC strains in the world, the use of global vaccines for ETEC infection is controversial. B subunit of heat labile toxin (LTB was introduced as a vaccine candidate molecule by several investigators. The expression of LTB gene isolated from a local bacterial strain and investigation of its immunological property was the objective of this study."nMaterials and Methods: LTB gene was isolated from a local isolated ETEC, cloned and expressed using pET28a expression vector. For LTB gene expression, the three main expression parameters (IPTG concentration, time and temperature of induction were investigated. The recombinant protein was purified ( > 95% with Ni-NTA column using 6XHis-tag and used as an antigen in ELISA test."nResults: The immunological analyses showed production of high titer of specific antibody in immunized mice. Anti LTB Antibody could bind to whole toxin and neutralize the toxin through inhibition of its binding to the Ganglioside M1 receptor."nConclusion: The recombinant LTB protein is a highly immunogenic molecule. Considering the LTB role in ETEC pathogenesis, it can be taken into account as one of the most important components of vaccines against local ETEC.

  17. Mouse mutants for the nicotinic acetylcholine receptor ß2 subunit display changes in cell adhesion and neurodegeneration response genes.

    Directory of Open Access Journals (Sweden)

    Carol M Rubin

    Full Text Available Mice lacking expression of the ß2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2 display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC axons to their dorsal lateral geniculate nuclei (dLGNs. Transcriptomes of LGN tissue from two independently generated Chrnb2-/- mutants and from wildtype mice were obtained at postnatal day 4 (P4, during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1, a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1 and chemokine (C-C motif ligand 21 (Ccl21 mRNAs in Chrnb2-/- mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2-/- mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2-/- mutant strains reveals the effects of genetic background upon gene expression.

  18. Mouse hippocampal GABAB1 but not GABAB2 subunit-containing receptor complex levels are paralleling retrieval in the multiple-T-maze

    Directory of Open Access Journals (Sweden)

    Soheil eKeihan Falsafi


    Full Text Available GABAB receptors are heterodimeric G-protein coupled receptors known to be involved in learning and memory. Although a role for GABAB receptors in cognitive processes is evident, there is no information on hippocampal GABAB receptor complexes in a multiple T maze (MTM task, a robust paradigm for evaluation of spatial learning.Trained or untrained (yoked control C57BL/6J male mice (n=10/group were subjected to the MTM task and sacrificed 6 hours following their performance. Hippocampi were taken, membrane proteins extracted and run on blue native PAGE followed by immunoblotting with specific antibodies against GABAB1, GABAB1a and GABAB2. Immunoprecipitation with subsequent mass spectrometric identification of co-precipitates was carried out to show if GABAB1 and GABAB2 as well as other interacting proteins co-precipitate. An antibody shift assay (ASA and a proximity ligation assay (PLA were also used to see if the two GABAB subunits are present in the receptor complex.Single bands were observed on Western blots, each representing GABAB1, GABAB1a or GABAB2 at an apparent molecular weight of approximately 100 kDa. Subsequently, densitometric analysis revealed that levels of GABAB1 and GABAB1a but not GABAB2- containing receptor complexes were significantly higher in trained than untrained groups. Immunoprecipitation followed by mass spectrometric studies confirmed the presence of GABAB1, GABAB2, calcium calmodulin kinases I and II, GluA1 and GluA2 as constituents of the complex. ASA and PLA also showed the presence of the two subunits of GABAB receptor within the complex. It is shown that increased levels of GABAB1 subunit-containing complexes are paralleling performance in a land maze.

  19. Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ are linked to hibernating state in hamsters

    Directory of Open Access Journals (Sweden)

    Alò Raffaella


    Full Text Available Abstract Background The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB and hibernating (HIB hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. Results Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p 1 ratio (over total α subunits considered in the present study in the medial preoptic area (MPOA and arcuate nucleus (Arc of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%. Regarding the other two subunits (β and γ, elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions. Conclusion We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional

  20. Identification of the binding subunit of the D/sub 1/-dopamine receptor by photoaffinity crosslinking

    Energy Technology Data Exchange (ETDEWEB)

    Amlaiky, N.; Berger, J.G.; Chang, W.; McQuade, R.D.; Caron, M.G.


    A derivative of the potent D/sub 1/ antagonist SCH-23390 has been synthesized. This compound, (R,S)-1-(m-aminophenyl)-7-chloro-8-hydroxy-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-38548), has been radioiodinated by a chloramine T procedure yielding 3 radioiodinated products. One of these separated isomers (R/sub f/ = 0.35; CH/sub 2/Cl/sub 2/:MEOH:TEA; 82.5:17.5:0.1 on TLC) binds reversibly to rat striatal membranes with high affinity (K/sub D/ approx. 80 pM) appropriate stereoselectivity and D/sub 1/-dopaminergic specificity. (/sup 125/I)SCH-38548 can be covalently incorporated into a peptide of M/sub r/ approx. 72,000 using the heterobifunctional crosslinking reagent N-succinimidyl-6-(4'-azido-2'-nitro-phenylamino) hexanoate. Covalent incorporation of (/sup 125/I) SCH-38548 into the M/sub r/ approx. 72,000 peptide can be blocked by dopaminergic agents with D/sub 1/-dopaminergic specificity (for agonists: SKF 38393 > apomorphine > dopamine; for antagonists: SCH-23390 > cis-flupentixol >>> trans-flupentixol). The D/sub 1/-dopaminergic selectivity and specificity of the labeling was further demonstrated by the fact that other antagonists such as domperidone, ketanserin, phentolamine and alprenolol did not compete for the covalent labeling of the M/sub r/ approx. 72,000 peptide. This new radioligand should be useful in the molecular characterization of the D/sub 1/-dopaminergic receptor from various sources.

  1. Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices. (United States)

    Small, D L; Poulter, M O; Buchan, A M; Morley, P


    Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-D-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions.

  2. Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

    DEFF Research Database (Denmark)

    Kristensen, Bjarne Winther; Noraberg, J; Zimmer, J


    ) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed...... by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity...

  3. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.;


    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  4. Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation. (United States)

    Jones, K L; Bagley, C J; Butcher, C; Barry, S C; Vadas, M A; D'Andrea, R J


    A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.

  5. Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

    DEFF Research Database (Denmark)

    Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan;


    responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27...

  6. Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures

    DEFF Research Database (Denmark)

    Bernardino, Liliana; Xapelli, Sara; Silva, Ana P


    The inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects...... of mouse recombinant TNF-alpha (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-alpha to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol....... By using TNF-alpha receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-alpha involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed...

  7. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

    Directory of Open Access Journals (Sweden)

    Sandra M McLachlan

    Full Text Available Transgenic mice with the human thyrotropin-receptor (TSHR A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors, regulatory T cell (Treg depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens

  8. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'. (United States)

    McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil


    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

  9. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABAA receptors on parvalbumin interneurons

    Directory of Open Access Journals (Sweden)

    Isabella eFerando


    Full Text Available Gamma (γ oscillations (30-120 Hz, an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs and the principal (pyramidal cells. PV+BCs express δ-subunit-containing GABAARs (-GABAARs and NMDA receptors (NMDA-Rs that balance the frequency of γ oscillations. Neuroactive steroids (NS, such as the progesterone-derived (3α,5α-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO, modulate the expression of δ-GABAARs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABAARs expression. Here we show that in CA3, where most PV+ interneurons (INs express δ-GABAARs, expression of δ-GABAARs on INs diminishes during pregnancy, but reverts to control levels within 48 hours postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABAARs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABAAR expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

  10. Ovarian cycle-linked plasticity of δ-GABAA receptor subunits in hippocampal interneurons affects γ oscillations in vivo

    Directory of Open Access Journals (Sweden)

    Albert Miklos Barth


    Full Text Available GABAA receptors containing δ subunits (δ-GABAARs are GABA-gated ion channels with extra- and perisynaptic localization, strong sensitivity to neurosteroids (NS, and a high degree of plasticity. In selective brain regions they are expressed on specific principal cells and interneurons (INs, and generate a tonic conductance that controls neuronal excitability and oscillations. Plasticity of δ-GABAARs in principal cells has been described during states of altered NS synthesis including acute stress, puberty, ovarian cycle, pregnancy and the postpartum period, with direct consequences on neuronal excitability and network dynamics. The defining network events implicated in cognitive function, memory formation and encoding are γ oscillations (30-120 Hz, a well-timed loop of excitation and inhibition between principal cells and PV-expressing INs (PV+INs. The δ-GABAARs of INs can modify γ oscillations, and a lower expression of δ-GABAARs on INs during pregnancy alters γ frequency recorded in vitro. The ovarian cycle is another physiological event with large fluctuations in NS levels and δ-GABAARs. Stages of the cycle are paralleled by swings in memory performance, cognitive function, and mood in both humans and rodents. Here we show δ-GABAARs changes during the mouse ovarian cycle in hippocampal cell types, with enhanced expression during diestrus in principal cells and specific INs. The plasticity of δ-GABAARs on PV-INs decreases the magnitude of γ oscillations continuously recorded in area CA1 throughout several days in vivo during diestrus and increases it during estrus. Such recurring changes in γ magnitude were not observed in non-cycling wild-type (WT females, cycling females lacking δ-GABAARs only on PV-INs (PV-Gabrd-/-, and in male mice during a time course equivalent to the ovarian cycle. Our findings may explain the impaired memory and cognitive performance experienced by women with premenstrual syndrome (PMS or premenstrual

  11. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABA(A) receptors on parvalbumin interneurons. (United States)

    Ferando, Isabella; Mody, Istvan


    Gamma (γ) oscillations (30-120 Hz), an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs) and the principal (pyramidal) cells. PV+BCs express δ-subunit-containing GABA(A)Rs (δ-GABA(A)Rs) and NMDA receptors (NMDA-Rs) that balance the frequency of γ oscillations. Neuroactive steroids (NS), such as the progesterone-derived (3α,5α)-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO), modulate the expression of δ-GABA(A)Rs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABA(A)Rs expression. Here we show that in CA3, where most PV+ interneurons (INs) express δ-GABA(A)Rs, expression of δ-GABA(A)Rs on INs diminishes during pregnancy, but reverts to control levels within 48 h postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy) may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABA(A)Rs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABA(A)R expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

  12. Regulation of epithelial sodium channel a-subunit expression by adenosine receptor A2a in alveolar epithelial cells

    Institute of Scientific and Technical Information of China (English)

    DENG Wang; WANG Dao-xin; ZHANG Wei; LI Chang-yi


    Background The amiloride-sensitive epithelial sodium channel a-subunit (a-ENaC) is an important factor for alveolar fluid clearance during acute lung injury. The relationship between adenosine receptor A2a (A2aAR) expressed in alveolar epithelial cells and aα-ENaC is poorly understood. We targeted the A2aAR in this study to investigate its role in the expression of αa-ENaC and in acute lung injury.Methods A549 cells were incubated with different concentrations of A2aAR agonist CGS-21680 and with 100 μmol/L CGS-21680 for various times. Rats were treated with lipopolysaccharide (LPS) after CGS-21680 was injected. Animals were sacrificed and tissue was harvested for evaluation of lung injury by analysis of the lung wet-to-dry weight ratio, lung permeability and myeloperoxidase activity. RT-PCR and Western blotting were used to determine the mRNA and protein expression levels of α-ENaC in A549 cells and alveolar type II epithelial cells.Results Both mRNA and protein levels of α-ENaC were markedly higher from 4 hours to 24 hours after exposure to 100μmol/L CGS-21680. There were significant changes from 0.1 umol/L to 100 μmol/L CGS-21680, with a positive correlation between increased concentrations of CGS-21680 and expression of α-ENaC. Treatment with CGS-21680during LPS induced lung injury protected the lung and promoted α-ENaC expression in the alveolar epithelial cells.Conclusion Activation of A2aAR has a protective effect during the lung injury, which may be beneficial to the prognosis of acute lung injury.

  13. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma. (United States)

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy


    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

  14. NMDA receptor subunits in the adult rat hippocampus undergo similar changes after 5 minutes in an open field and after LTP induction.

    Directory of Open Access Journals (Sweden)

    Maria Veronica Baez

    Full Text Available NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1 adult rats that had explored an open field (OF for 5 minutes, a time sufficient to induce habituation, 2 mature rat hippocampal neuron cultures depolarized by KCl and 3 hippocampal slices from adult rats where long term potentiation (LTP was induced by theta-burst stimulation (TBS. GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes--but not 30 minutes--after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor or actinomycin D (a transcription inhibitor during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.

  15. Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors

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    Marisa S Goo


    Full Text Available Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion or removal of AMPA-type glutamate receptors at the postsynaptic membrane of excitatory synapses. Once internalized, these receptors may be recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Alternatively, these receptors may be targeted for destruction by multiple degradation pathways in the cell. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. In this review, we will discuss recent findings on the role of ubiquitination in the trafficking and turnover of ionotropic glutamate receptors and plasticity of excitatory synapses.

  16. GABA-A and NMDA receptor subunit mRNA expression is altered in the caudate but not the putamen of the postmortem brains of alcoholics. (United States)

    Bhandage, Amol K; Jin, Zhe; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis


    Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here, we have studied the mRNA expression of ion channel receptors for glutamate and GABA in the dorsal striatum of post-mortem brains from alcoholics (n = 29) and normal controls (n = 29), with the focus on the caudate nucleus that is associated with the frontal cortex executive functions and automatic thinking and on the putamen area that is linked to motor cortices and automatic movements. The results obtained by qPCR assay revealed significant changes in the expression of specific excitatory ionotropic glutamate and inhibitory GABA-A receptor subunit genes in the caudate but not the putamen. Thus, in the caudate we found reduced levels of mRNAs encoding the GluN2A glutamate receptor and the δ, ε, and ρ2 GABA-A receptor subunits, and increased levels of the mRNAs encoding GluD1, GluD2, and GABA-A γ1 subunits in the alcoholics as compared to controls. Interestingly in the controls, 11 glutamate and 5 GABA-A receptor genes were more prominently expressed in the caudate than the putamen (fold-increase varied from 1.24 to 2.91). Differences in gene expression patterns between the striatal regions may underlie differences in associated behavioral outputs. Our results suggest an altered balance between caudate-mediated voluntarily controlled and automatic behaviors in alcoholics, including diminished executive control on goal-directed alcohol-seeking behavior.

  17. Ahsg-fetuin blocks the metabolic arm of insulin action through its interaction with the 95-kD β-subunit of the insulin receptor. (United States)

    Goustin, Anton Scott; Derar, Nada; Abou-Samra, Abdul B


    We previously have shown that Ahsg, a liver glycoprotein, inhibits insulin receptor (InsR) tyrosine kinase (TK) activity and the ERK1/2 mitogenic signaling arm of insulin signaling. Here we show that Ahsg blocks insulin-stimulated GLUT4 translocation and Akt activation in intact cells (mouse myoblasts). Furthermore, Ahsg inhibits InsR autophosphorylation of highly-purified insulin holoreceptors in a cell-free, ATP-dependent system, with an IC50 within the range of single-chain Ahsg concentrations in human serum. Binding of (125)I-insulin to living cells overexpressing the InsR shows a dissociation constant (KD) of 250pM, unaltered in the presence of 300 nM Ahsg. A mutant InsR cDNA encoding the signal peptide, the β-subunit and the furin processing site, but deleting the α-subunit, was stably expressed in HEK293 cells. Treatment with peroxovanadate, but not insulin, dramatically increased the 95 kD β-subunit tyrosine phosphoryation. The level of tyrosine phosphorylation of the 95-kD β-subunit can be driven down sharply by treatment of living HEK293 transfectant cells with physiological doses of Ahsg. Treatment of myogenic cells with Ahsg blunts insulin-stimulated InsR autophosphorylation and AKT phosphorylation. Taken together, we show that Ahsg antagonizes the metabolic functions initiated by InsR activation without interference in insulin binding. The experiments suggest a direct interaction of Ahsg with the InsR ectodomain β-subunit in a mode that does not significantly alter the high-affinity binding of insulin to the holoreceptor's two complementing α-subunits.

  18. N-methyl-D-aspartate receptor NR2B subunit involved in depression-like behaviours in lithium chloride-pilocarpine chronic rat epilepsy model. (United States)

    Peng, Wei-Feng; Ding, Jing; Li, Xin; Fan, Fan; Zhang, Qian-Qian; Wang, Xin


    Depression is a common comorbidity in patients with epilepsy with unclear mechanisms. This study is to explore the role of glutamate N-methyl-D-aspartate (NMDA) receptor NR1, NR2A and NR2B subunits in epilepsy-associated depression. Lithium chloride (Licl)-pilocarpine chronic rat epilepsy model was established and rats were divided into epilepsy with depression (EWD) and epilepsy without depression (EWND) subgroups based on forced swim test. Expression of NMDA receptor NR1, NR2A and NR2B subunits was measured by western blot and immunofluorescence methods. The immobility time (IMT) was significantly greater in Licl-pilocarpine model group than in Control group, which was also greater in EWD group than in EWND group. No differences of spontaneous recurrent seizure (SRS) counts over two weeks and latency were found between EWD and EWND groups. The number of NeuN positive cells was significantly less in Licl-pilocarpine model group than in Control group, but had no difference between EWD and EWND groups. The ratios of phosphorylated NR1 (p-NR1)/NR1 and p-NR2B/NR2B were significantly greater in the hippocampus in EWD group than in EWND group. Moreover, the expression of p-NR1 and p-NR2B in the CA1 subfield of hippocampus were both greater in Licl-pilocarpine model group than Control group. Selective blockage of NR2B subunit with ifenprodil could alleviate depression-like behaviours of Licl-pilocarpine rat epilepsy model. In conclusion, glutamate NMDA receptor NR2B subunit was involved in promoting depression-like behaviours in the Licl-pilocarpine chronic rat epilepsy model and might be a target for treating epilepsy-associated depression.

  19. Effects of Traumatic Stress Induced in the Juvenile Period on the Expression of Gamma-Aminobutyric Acid Receptor Type A Subunits in Adult Rat Brain

    Directory of Open Access Journals (Sweden)

    Cui Yan Lu


    Full Text Available Studies have found that early traumatic experience significantly increases the risk of posttraumatic stress disorder (PTSD. Gamma-aminobutyric acid (GABA deficits were proposed to be implicated in development of PTSD, but the alterations of GABA receptor A (GABAAR subunits induced by early traumatic stress have not been fully elucidated. Furthermore, previous studies suggested that exercise could be more effective than medications in reducing severity of anxiety and depression but the mechanism is unclear. This study used inescapable foot-shock to induce PTSD in juvenile rats and examined their emotional changes using open-field test and elevated plus maze, memory changes using Morris water maze, and the expression of GABAAR subunits (γ2, α2, and α5 in subregions of the brain in the adulthood using western blotting and immunohistochemistry. We aimed to observe the role of GABAAR subunits changes induced by juvenile trauma in the pathogenesis of subsequent PTSD in adulthood. In addition, we investigated the protective effects of exercise for 6 weeks and benzodiazepine (clonazepam for 2 weeks. This study found that juvenile traumatic stress induced chronic anxiety and spatial memory loss and reduced expression of GABAAR subunits in the adult rat brains. Furthermore, exercise led to significant improvement as compared to short-term BZ treatment.

  20. Expression of mRNA-encoding subunits of N-methyl-D-aspartate receptor in the hypothalamus in sustained monaural block of auditory air-conduction model rats

    Institute of Scientific and Technical Information of China (English)

    Ping Wan; Xiaojian Lai; Cheng Huang; Xinde Sun


    A sustained monaural block of auditory air-conduction model was established in rats through subcutaneous suture in the right ear canal. The gene expression levels of hypothalamic N-methyl-D-aspartate receptor NR1, NR2A, NR2B and NR2C mRNA in the auditory central nervous system of Sprague-Dawley rats at postnatal 9, 23, 37 days were determined after an environmental change. Reverse transcription-PCR assay showed that the critical period for the development of NR1, NR2A, and NR2B subunits in the left hypothalamus and NR1- and NR2B-dependent auditory neurons in the right hypothalamus terminated 23 days after the suture in the right ear. The critical period for the development of NR2A subunit-dependent auditory neurons in the right hypothalamus was terminated by postnatal day 37. The results confirmed that N-methyl-D-aspartate receptor subunits in the hypothalamus may be regulated by the auditory environment.

  1. The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

    Directory of Open Access Journals (Sweden)

    Sally M Williamson


    Full Text Available Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect approximately 1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5:1 (Asu-unc-38ratioAsu-unc-29, nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1:5 Asu-unc-38ratioAsu-unc-29, levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the

  2. 丙泊酚对大鼠胶质瘤细胞侵袭和迁移能力的影响及ADAR2-AMPA受体GluR2通路在其中的作用%Effects of propofol on invasion and migration of glioma cells in rats and the role of ADAR2-AMPA receptor GluR2 pathway

    Institute of Scientific and Technical Information of China (English)

    王欣悦; 王海云; 王国林; 杨卓; 张涛


    目的 评价丙泊酚对大鼠胶质瘤细胞侵袭和迁移能力的影响及腺苷脱氨酶(ADAR2)-α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体GluR2通路在其中的作用.方法 传代培养大鼠C6胶质瘤细胞,采用随机数字表法分为4组(n=24):对照组(C组)、丙泊酚组(P组)、阴性siRNA转染+丙泊酚组(NP组)和ADAR2-siRNA转染+丙泊酚组(AP组).C组正常培养;NP组和AP组分别将阴性siRNA或ADAR2-siRNA转染至细胞内,48 h后处理同P组;P组加入丙泊酚,终浓度1.2 μg/ml,孵育6h后换为正常培养液,继续培养18h.采用MTT比色分析法检测细胞活力,Transwell侵袭实验测定侵袭细胞数,细胞划痕实验测定迁移率,Western blot法检测胞核ADAR2和胞膜GluR2的表达.结果 与C组比较,P组和NP组细胞活力、侵袭细胞数和迁移率降低,胞核A-DAR2及胞膜GluR2表达上调(P<0.05);与P组比较,AP组细胞活力、侵袭细胞数和迁移率升高,胞核ADAR2及胞膜GluR2表达下调(P<0.05);与NP组比较,AP组细胞活力、侵袭细胞数和迁移率升高,胞核ADAR2及胞膜GluR2表达下调(P<0.05).结论 丙泊酚可抑制大鼠胶质瘤细胞的侵袭和迁移能力,其机制与激活ADAR2-AMPA受体GluR2通路有关.%Objective To evaluate the effects of propofol on the invasion and migration of glioma cells in the rats and the role of adenosine deaminase acting on RNA 2 (ADAR2)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit glutamate 2 (GluR2) pathway.Methods C6 glioma cells were subcuhured and randomly divided into 4 groups (n =24 each) using a random number table:control group (group C);propofol group (group P);negative siRNA transfection + propofol group (group NP);ADAR2-siRNA transfection + propofol group (group AP).The cells were cultured in the common culture medium in group C.In NP and AP groups,negative siRNA and ADAR2-siRNA were transfected into the cells,respectively,and 48 h later the other procedures were similar

  3. NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801. (United States)

    Roshanravan, Hila; Kim, Eun Young; Dryer, Stuart E


    N-methyl-d-aspartate (NMDA) receptors are expressed throughout the kidney, and the abundance of these receptors and some of their endogenous agonists are increased in diabetes. Moreover, sustained activation of podocyte NMDA receptors induces Ca(2+) influx, oxidative stress, loss of slit diaphragm proteins, and apoptosis. We observed that NMDA receptor subunits and their transcripts are increased in podocytes and mesangial cells cultured in elevated glucose compared with controls. A similar increase in NMDA subunits, especially NR1, NR2A, and NR2C, was observed in glomeruli and tubules of Akita mice. Sustained continuous treatment with the strong NMDA receptor antagonist dizocilpine (MK-801) for 28 days starting at 8 weeks of age reduced 24-h albumin excretion and mesangial matrix expansion and improved glomerular ultrastructure in Akita mice. MK-801 did not alleviate reduced Akita mouse body weight and had no effect on kidney histology or ultrastructure in DBA/2J controls. The structurally dissimilar NMDA antagonist memantine also reduced diabetic nephropathy, although it was less effective than MK-801. Inhibition of NMDA receptors may represent a valid therapeutic approach to reduce renal complications of diabetes, and it is possible to develop well-tolerated agents with minimal central nervous system effects. Two such agents, memantine and dextromethorphan, are already in widespread clinical use.

  4. Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET of Dihydropyridine Receptor (DHPR β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

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    Dipankar Bhattacharya

    Full Text Available The dihydropyridine receptor (DHPR β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1 complex is still debatable. We used fluorescence resonance energy transfer (FRET to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1 myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

  5. Treatment with dehydroepiandrosterone sulfate increases NR1 and NR2 subunits of NMDA receptors in rat hippocampus.

    Institute of Scientific and Technical Information of China (English)



    Neurosteroids are present in the central nervous system (CNS) and can be allosteric modulators of neurotransmitter receptors. One of them, the dehydroepiandrosterone sulfate (DHEAS) has been shown to modulate the NMDA receptor, a subtype of glutamate receptor. These NMDA receptors are known to be involved in long-term potentiation and in learning and memory (Tsien 2000). Moreover, DHEAS has been reported

  6. Object-in-place associative recognition memory depends on glutamate receptor neurotransmission within two defined hippocampal-cortical circuits: a critical role for AMPA and NMDA receptors in the hippocampus, perirhinal, and prefrontal cortices. (United States)

    Barker, Gareth Robert Issac; Warburton, Elizabeth Clea


    Object-in-place associative recognition memory depends on an interaction between the hippocampus (HPC), perirhinal (PRH), and medial prefrontal (mPFC) cortices, yet the contribution of glutamate receptor neurotransmission to these interactions is unknown. NMDA receptors (NMDAR) in the HPC were critical for encoding of object-in-place memory but not for single-item object recognition. Next, a disconnection procedure was used to examine the importance of "concurrent" glutamate neurotransmission in the HPC-mPFC and HPC-PRH. Contralateral unilateral infusions of NBQX (AMPAR antagonist), into the HPC-mPFC, or HPC-PRH, either before acquisition or test, impaired object-in-place performance. Thus, both circuits are necessary for encoding and retrieval. Crossed unilateral AP5 (NMDAR antagonist) infusions into the HPC-mPFC or HPC-PRH impaired encoding, but not retrieval. Specifically crossed HPC-mPFC infusions impaired both short-term (5 min) and longer term (1 h) memory while HPC-PRH infusions impaired longer term memory only. This delay-dependent effect of AP5 in the HPC-PRH on object-in-place memory, accords with its effects in the PRH, on single item object recognition memory, thereby suggesting that a single PRH synaptic plasticity mechanism underpins different recognition memory processes. Further, blocking excitatory neurotransmission in any pair of structures within the networks impaired "both" encoding and retrieval, thus object-in-place memory clearly requires network interdependency across multiple structures.

  7. Expression of gamma-aminobutyric acid A receptor subunits α1,β1,γ2 mRNA in rats with hepatic encephalopathy

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qing Li; Lei Dong; Zhong-Hua Liu; Jin-Yan Luo


    AIM To investigate the mRNA expression of gammaaminobutyric acid A (GABAA) receptor subunits α1,β1, γ2in different parts of the brain of rats with hepaticencephalopathy.METHODS: Twelve adult male Sprague-Dawley rats were randomly divided into two groups: (1) hepatic encephalopathy model group (n = 6), which was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg/kg) for threeconsecutive days; (2) control group (n = 6), in which the rats were treated with same dose of normal saline solution. After the freeze slice of cerebrum was made,in situ hybridization was used to detect the mRNA of GABAA receptor subunits α1, β1, and γ2 in rat cerebral cortex, basal nuclei, substantia nigra and hippocampi. Image data were collected and analyzed quantitatively by QWin550CWmodel image signal gather and analysis system. RESULTS: In rats with hepatic encephalopathy, mRNA expression levels of GABAA receptor subunits α1, β1 increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis and hippocampi (144.7±15.67/184.14±4.41, 60.61±33.66/113.07±32.44,87.71± 21.25/128.40±18.85, 122.34±5.56/161.60±4.56,123.29±5.21/140.65±4.15, 123.40±4.42/140.09±4.52,124.76±4.18/140.09±4.12, 141.62±15.09/182.80 ±5.20,69.13±30.74/134.21±43.76, 87.87±25.16/151.01±19.49,122.14±6.30/162.33±3.92, 122.81±5.09/137.19±7.12,123.00±4.63/138.11±5.92, 125.75 ±2.43/138.81±6.10,P<0.01), but did not change in the cerebral cortex compared to the control group. Similar changes were found in the mRNA expression levels of GABAA receptor subunit γ2,which increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis (136.81±26.41/167.97±16.23, 51.00±36.14/113.18±36.52, 86.35±20.30/126.90±19.74, P<0.01), CA1 of hippocampal (162.15±9.05/178.62±6.45, P<0.05), and no changes were found in the cerebral cortex and CA2, CA3, CA4 of hippocampi.CONCLUSION: In rats with hepatic

  8. MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit.

    Directory of Open Access Journals (Sweden)

    Robert A Volkmann

    Full Text Available GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenylsulfonamidomethyl-N-((2-methylthiazol-5-ylmethylpyrazine-2-carboxamide and MPX-007 (5-(((3-fluoro-4-fluorophenylsulfonamidomethyl-N-((2-methylthiazol-5-ylmethylmethylpyrazine-2-carboxamide. MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC50s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.

  9. MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit. (United States)

    Volkmann, Robert A; Fanger, Christopher M; Anderson, David R; Sirivolu, Venkata Ramana; Paschetto, Kathy; Gordon, Earl; Virginio, Caterina; Gleyzes, Melanie; Buisson, Bruno; Steidl, Esther; Mierau, Susanna B; Fagiolini, Michela; Menniti, Frank S


    GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC50s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.

  10. Repeated cocaine enhances ventral hippocampal-stimulated dopamine efflux in the nucleus accumbens and alters ventral hippocampal NMDA receptor subunit expression. (United States)

    Barr, Jeffrey L; Forster, Gina L; Unterwald, Ellen M


    Dopaminergic neurotransmission in the nucleus accumbens is important for various reward-related cognitive processes including reinforcement learning. Repeated cocaine enhances hippocampal synaptic plasticity, and phasic elevations of accumbal dopamine evoked by unconditioned stimuli are dependent on impulse flow from the ventral hippocampus. Therefore, sensitized hippocampal activity may be one mechanism by which drugs of abuse enhance limbic dopaminergic activity. In this study, in vivo microdialysis in freely moving adult male Sprague-Dawley rats was used to investigate the effect of repeated cocaine on ventral hippocampus-mediated dopaminergic transmission within the medial shell of the nucleus accumbens. Following seven daily injections of saline or cocaine (20 mg/kg, ip), unilateral infusion of N-methyl-d-aspartate (NMDA, 0.5 μg) into the ventral hippocampus transiently increased both motoric activity and ipsilateral dopamine efflux in the medial shell of the nucleus accumbens, and this effect was greater in rats that received repeated cocaine compared to controls that received repeated saline. In addition, repeated cocaine altered NMDA receptor subunit expression in the ventral hippocampus, reducing the NR2A : NR2B subunit ratio. Together, these results suggest that repeated exposure to cocaine produces maladaptive ventral hippocampal-nucleus accumbens communication, in part through changes in glutamate receptor composition. A behaviorally sensitizing regimen of cocaine (20 mg/kg, ip 7 days) also sensitized ventral hippocampus (hipp)-mediated dopaminergic transmission within the nucleus accumbens (Nac) to NMDA stimulation (bolts). This was associated with reduced ventral hippocampal NR2A:NR2B subunit ratio, suggesting that repeated exposure to cocaine produces changes in hippocampal NMDA receptor composition that lead to enhanced ventral hippocampus-nucleus accumbens communication.

  11. Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes. (United States)

    Scibek, Jeffery J; Evergren, Emma; Zahn, Stefan; Canziani, Gabriela A; Van Ryk, Donald; Chaiken, Irwin M


    To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

  12. Differential expression of glutamate receptors in avian neural pathways for learned vocalization. (United States)

    Wada, Kazuhiro; Sakaguchi, Hironobu; Jarvis, Erich D; Hagiwara, Masatoshi


    Learned vocalization, the substrate for human language, is a rare trait. It is found in three distantly related groups of birds-parrots, hummingbirds, and songbirds. These three groups contain cerebral vocal nuclei for learned vocalization not found in their more closely related vocal nonlearning relatives. Here, we cloned 21 receptor subunits/subtypes of all four glutamate receptor families (AMPA, kainate, NMDA, and metabotropic) and examined their expression in vocal nuclei of songbirds. We also examined expression of a subset of these receptors in vocal nuclei of hummingbirds and parrots, as well as in the brains of dove species as examples of close vocal nonlearning relatives. Among the 21 subunits/subtypes, 19 showed higher and/or lower prominent differential expression in songbird vocal nuclei relative to the surrounding brain subdivisions in which the vocal nuclei are located. This included relatively lower levels of all four AMPA subunits in lMAN, strikingly higher levels of the kainite subunit GluR5 in the robust nucleus of the arcopallium (RA), higher and lower levels respectively of the NMDA subunits NR2A and NR2B in most vocal nuclei and lower levels of the metabotropic group I subtypes (mGluR1 and -5) in most vocal nuclei and the group II subtype (mGluR2), showing a unique expression pattern of very low levels in RA and very high levels in HVC. The splice variants of AMPA subunits showed further differential expression in vocal nuclei. Some of the receptor subunits/subtypes also showed differential expression in hummingbird and parrot vocal nuclei. The magnitude of differential expression in vocal nuclei of all three vocal learners was unique compared with the smaller magnitude of differences found for nonvocal areas of vocal learners and vocal nonlearners. Our results suggest that evolution of vocal learning was accompanied by differential expression of a conserved gene family for synaptic transmission and plasticity in vocal nuclei. They also suggest

  13. Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

    DEFF Research Database (Denmark)

    Wang, Qian; Pless, Stephan Alexander; Lynch, Joseph W


    Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational...... changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements...... from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting...

  14. Structural determinants within residues 180-199 of the rodent. alpha. 5 nicotinic acetylcholine receptor subunit involved in. alpha. -bungarotoxin binding

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    McLane, K.E.; Xiadong Wu; Conti-Tronconi, B.M. (Univ. of Minnesota, St. Paul (United States))


    Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) {alpha} subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. The authors have previously defined {alpha}-bungarotoxin ({alpha}-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR {alpha} subunit, designated {alpha}5, and between residues 181 and 200 of the chick neuronal {alpha}7 and {alpha}8 subunits. These sequences are relatively divergent compared with the Torpedo and muscle nAChR {alpha}1 {alpha}-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the {alpha}5 sequence, they were interested in determining the critical amino acid residues for {alpha}-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat {alpha}(180-199) sequence were tested, using a competition assay, in which peptides compete for {sup 125}I-{alpha}-BTX binding with native Torpedo nAChR. These results indicate that a disulfide bridge between the vicinal cysteines at positions 191 and 192 of the {alpha}5 sequence is not an absolute requirement for {alpha}-BTX binding activity.

  15. Dopamine D3 receptor-dependent changes in alpha6 GABAA subunit expression in striatum modulate anxiety-like behaviour: Responsiveness and tolerance to diazepam. (United States)

    Leggio, Gian Marco; Torrisi, Sebastiano Alfio; Castorina, Alessandro; Platania, Chiara Bianca Maria; Impellizzeri, Agata Antonia Rita; Fidilio, Annamaria; Caraci, Filippo; Bucolo, Claudio; Drago, Filippo; Salomone, Salvatore


    Increasing evidence indicates that central dopamine (DA) neurotransmission is involved in pathophysiology of anxiety, in particular the DA receptor subtype 3 (D3R). We previously reported that D3R null mice (D3R(-/-)) exhibit low baseline anxiety levels and that acutely administrated diazepam is more effective in D3R(-/-) than in wild type (WT) when tested in the elevated plus maze test (EPM). Here we tested the hypothesis that genetic deletion or pharmacological blockade of D3R affect GABAA subunit expression, which in turn modulates anxiety-like behaviour as well as responsiveness and tolerance to diazepam. D3R(-/-) mice exhibited tolerance to diazepam (0.5mg/kg, i.p.), assessed by EPM, as fast as after 3 day-treatment, performing similarly to untreated D3R(-/-) mice; conversely, WT exhibited tolerance to diazepam after a 14-21 day-treatment. Analysis of GABAA α6 subunit mRNA expression by qPCR in striatum showed that it was about 15-fold higher in D3R(-/-) than in WT. Diazepam treatment did not modify α6 expression in D3R(-/-), but progressively increased α6 expression in WT, to the level of untreated D3R(-/-) after 14-21 day-treatment. BDNF mRNA expression in striatum was remarkably (>10-fold) increased after 3 days of diazepam-treatment in both WT and D3R(-/-); such expression level, however, slowly declined below control levels, by 14-21 days. Following a 7 day-treatment with the selective D3R antagonist SB277011A, WT exhibited a fast tolerance to diazepam accompanied by a robust increase in α6 subunit expression. In conclusion, genetic deletion or pharmacological blockade of D3R accelerate the development of tolerance to repeated administrations of diazepam and increase α6 subunit expression, a GABAA subunit that has been linked to diazepam insensitivity. Modulation of GABAA receptor by DA transmission may be involved in the mechanisms of anxiety and, if occurring in humans, may have therapeutic relevance following repeated use of drugs targeting D3R.

  16. Expression of 10 GABA(A) receptor subunit messenger RNAs in the motor-related thalamic nuclei and basal ganglia of Macaca mulatta studied with in situ hybridization histochemistry. (United States)

    Kultas-Ilinsky, K; Leontiev, V; Whiting, P J


    In situ hybridization histochemistry technique with [35S]UTP-labelled riboprobes was used to study the expression pattern of 10 GABA(A) receptor subunit messenger RNAs in the basal ganglia and motor thalamic nuclei of rhesus monkey. Human transcripts were used for the synthesis of alpha2, alpha4, beta2, beta3, gamma1 and delta subunit messenger RNA probes. Rat complementary DNAs were used for generating alpha1, alpha3, beta1 and gamma2 subunit messenger RNA probes. Nigral, pallidal and cerebellar afferent territories in the ventral tier thalamic nuclei all expressed alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, delta and gamma2 subunit messenger RNAs but at different levels. Each intralaminar nucleus displayed its own unique expression pattern. In the thalamus, gamma1 subunit messenger RNA was detected only in the parafascicular nucleus. Comparison of the expression patterns with the known organization of GABA(A) connections in thalamic nuclei suggests that (i) the composition of the receptor associated with reticulothalamic synapses, except for those in the intralaminar nuclei, may be alpha1alpha4beta2delta, (ii) receptors of various other subunit compositions may operate in the local GABAergic circuits, and (iii) the composition of receptors at nigro- and pallidothalamic synapses may differ, with those at nigrothalamic probably containing beta1 and gamma2 subunits. In the medial and lateral parts of the globus pallidus, the subthalamic nucleus and the substantia nigra pars reticularis, the alpha1, beta2 and gamma2 messenger RNAs were co-expressed at a high level suggesting that this subunit composition was associated with all GABAergic synapses in the direct and indirect striatal output pathways. Various other subunit messenger RNAs were also expressed but at a lower level. In the substantia nigra pars compacta the most highly expressed messenger RNAs were alpha3, alpha4 and beta3; all other subunit messenger RNAs studied, except for gamma1, alpha1 and

  17. Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy. (United States)

    Bouilleret, V; Loup, F; Kiener, T; Marescaux, C; Fritschy, J M


    Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of

  18. The subunit gene Ldα1 of nicotinic acetylcholine receptors plays important roles in the toxicity of imidacloprid and thiamethoxam against Leptinotarsa decemlineata. (United States)

    Qu, Yang; Chen, Jinhua; Li, Chenge; Wang, Qiang; Guo, Wenchao; Han, Zhaojun; Jiang, Weihua


    Nicotinic acetylcholine receptors (nAChRs) are pentameric ACh-gated ion channels. It is believed that nAChRs composed of different subunits may vary in their function and toxicological characteristics. Neonicotinoids are activators of nAChRs and important insecticides that are extensively used for crop protection and resistance has been developed by some pests. They are also major insecticides for the control of Leptinotarsa decemlineata, which is a destructive defoliator pest that invaded the Xinjiang region of China in the 1990s. However, little is known about the constitution or subunits of the target in this pest. In this study, the full-length cDNAs encoding four new nAChR subunits (named Ldα3, Ldα6, Ldα10, and Ldβ1) were cloned from L. decemlineata. These genes encode 822-, 753-, 672-, and 759-amino acid proteins, respectively, which share typical features of insect nAChRs subunits and closely resemble the corresponding subunits of the nAChRs from Tribolium castaneum. Temporal and spatial expression analyses showed that these genes, as well as the previously identified Ldα1, Ldα2, and Ldα8 genes, are widely expressed in all developmental stages, including eggs, larvae of various instars, pupae, and adults. All genes monitored were expressed at higher levels in the head than in the thorax and abdomen, except for Ldα10. Dietary ingestion of double-stranded RNA bacterially expressed for Ldα1 (dsLdα1) significantly reduced the mRNA level of Ldα1 in treated larvae and adults by 48.0% and 78.6%, respectively. Among the non-target genes, Ldα3, Ldα9, and Ldβ1 were significantly up-regulated in larvae. A toxicity bioassay showed that dsLdα1 treatment greatly decreased the sensitivity to imidacloprid and thiamethoxam in adults. The larval susceptibility to thiamethoxam but not to imidacloprid was also reduced because of the lower down-regulation of Ldα1. Thus, our results suggest that Ldα1 encodes a subunit of a functional nAChR that mediates the

  19. GluN1 and GluN2A NMDA Receptor Subunits Increase in the Hippocampus during Memory Consolidation in the Rat (United States)

    Cercato, Magali C.; Vázquez, Cecilia A.; Kornisiuk, Edgar; Aguirre, Alejandra I.; Colettis, Natalia; Snitcofsky, Marina; Jerusalinsky, Diana A.; Baez, María V.


    It is widely accepted that NMDA receptors (NMDAR) are required for learning and memory formation, and for synaptic plasticity induction. We have previously shown that hippocampal GluN1 and GluN2A NMDAR subunits significantly increased following habituation of rats to an open field (OF), while GluN2B remained unchanged. Similar results were obtained after CA1-long-term potentiation (LTP) induction in rat hippocampal slices. Other studies have also shown NMDAR up regulation at earlier and later time points after LTP induction or learning acquisition. In this work, we have studied NMDAR subunits levels in the hippocampus and prefrontal cortex (PFC) after OF habituation and after object recognition (OR), to find out whether rising of NMDAR subunits is a general and structure-specific feature during memory formation. In 1, 2 and 3 month old rats there was an increase in hippocampal GluN1 and GluN2A, but not in GluN2B levels 70 min after OF habituation. This rise overlaps with early phase of memory consolidation, suggesting a putative relationship between them. The increases fell down to control levels 90 min after training. Similar results were obtained in the hippocampus of adult rats 70 min after OR training, without changes in PFC. Following OF test or OR discrimination phase, NMDAR subunits remained unchanged. Hence, rising of hippocampal GluN1 and GluN2A appears to be a general feature after novel “spatial/discrimination” memory acquisition. To start investigating the dynamics and possible mechanisms of these changes, we have studied hippocampal neuron cultures stimulated by KCl to induce plasticity. GluN1 and GluN2A increased both in dendrites and neuronal bodies, reaching a maximum 75 min later and returning to control levels at 90 min. Translation and/or transcription and mobilization differentially contribute to this rise in subunits in bodies and dendrites. Our results showed that the NMDAR subunits increase follows a similar time course both in vitro and

  20. Excitatory synapses on dendritic shafts of the caudal basal amygdala exhibit elevated levels of GABAA receptor α4 subunits following the induction of activity-based anorexia. (United States)

    Wable, Gauri S; Barbarich-Marsteller, Nicole C; Chowdhury, Tara G; Sabaliauskas, Nicole A; Farb, Claudia R; Aoki, Chiye


    Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation, excessive exercise, and anxiety. The onset of AN is most often at puberty, suggesting that gonadal hormonal fluctuations may contribute to AN vulnerability. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction. The basal amygdala as well as the GABAergic system regulate trait anxiety. We therefore examined the subcellular distribution of GABA receptors (GABARs) in the basal amygdala of female pubertal rats and specifically of their α4 subunits, because expression of α4-containing GABARs is regulated by gonadal hormone fluctuations. Moreover, because these GABARs reduce neuronal excitability through shunting of EPSPs, we quantified the frequency of occurrence of these GABARs adjacent to excitatory synapses. Electron microscopic immunoctychemistry revealed no change in the frequency of association of α4 subunits with excitatory synapses on dendritic spines, whether in the anterior (Bregma -2.8 mm) or caudal (Bregma -3.8 mm) portion of the basal amygdala. Sholl analysis of golgi-stained neurons also revealed no change in the extent of dendritic branching by these densely spiny, pyramidal-like neurons. However, there was an increase of membranous α4 subunits near excitatory synapses on dendritic shafts, specifically in the caudal basal amygdala, and this was accompanied by a rise of α4 subunits intracellularly. Because most dendritic shafts exhibiting excitatory synapses are GABAergic interneurons, the results predict disinhibition, which would increase excitability of the amygdaloid network, in turn augmenting ABA animals' anxiety.

  1. Dizocilpine (MK-801) induces distinct changes of N-methyl-D-aspartic acid receptor subunits in parvalbumin-containing interneurons in young adult rat prefrontal cortex. (United States)

    Xi, Dong; Zhang, Wentong; Wang, Huai-Xing; Stradtman, George G; Gao, Wen-Jun


    N-methyl-D-aspartic acid receptor (NMDAR) hypofunction has long been implicated in schizophrenia and NMDARs on gamma-aminobutyric acid (GABA)ergic interneurons are proposed to play an essential role in the pathogenesis. However, controversial results have been reported regarding the regulation of NMDAR expression, and direct evidence of how NMDAR antagonists act on specific subpopulations of prefrontal interneurons is missing. We investigated the effects of the NMDAR antagonist dizocilpine (MK-801) on the expression of NMDAR subtypes in the identified interneurons in young adult rat prefrontal cortex (PFC) by using laser microdissection and real-time polymerase chain reaction, combined with Western blotting and immunofluorescent staining. We found that MK-801 induced distinct changes of NMDAR subunits in the parvalbumin-immunoreactive (PV-ir) interneurons vs. pyramidal neurons in the PFC circuitry. The messenger RNA (mRNA) expression of all NMDAR subtypes, including NR1 and NR2A to 2D, exhibited inverted-U dose-dependent changes in response to MK-801 treatment in the PFC. In contrast, subunit mRNAs of NMDARs in PV-ir interneurons were significantly down-regulated at low doses, unaltered at medium doses, and significantly decreased again at high doses, suggesting a biphasic dose response to MK-801. The differential effects of MK-801 in mRNA expression of NMDAR subunits were consistent with the protein expression of NR2A and NR2B subunits revealed with Western blotting and double immunofluorescent staining. These results suggest that PV-containing interneurons in the PFC exhibit a distinct responsiveness to NMDAR antagonism and that NMDA antagonist can differentially and dose-dependently regulate the functions of pyramidal neurons and GABAergic interneurons in the prefrontal cortical circuitry.

  2. Involvement of β3A Subunit of Adaptor Protein-3 in Intracellular Trafficking of Receptor-like Protein Tyrosine Phosphatase PCP-2

    Institute of Scientific and Technical Information of China (English)

    Hui DONG; Hong YUAN; Weirong JIN; Yan SHEN; Xiaojing XU; Hongyang WANG


    PCP-2 is a human receptor-like protein tyrosine phosphatase and a member of the MAM domain family cloned in human pancreatic adenocarcinoma cells. Previous studies showed that PCP-2 directly interacted with β-catenin through the juxtamembrane domain, dephosphorylated β-catenin and played an important role in the regulation of cell adhesion. Recent study showed that PCP-2 was also involved in the repression of β-catenin-induced transcriptional activity. Here we describe the interactions of PCP-2 with the β3A subunit of adaptor protein (AP)-3 and sorting nexin (SNX) 3. These protein complexes were detected using the yeast two-hybrid assay with the juxtamembrane and membrane-proximal catalytic domain of PCP-2 as "bait". Both AP-3 and SNX3 are molecules invol