Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Eq, R, M, H||IP, WB, IH(P)||Rb||Affinity Purified||Polyclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at 2-8°C from date of receipt.|
|Material Size||200 µL|
References | 51 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Select 3',5'-cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brain. |
Kelly, Michy P, et al.
Cell. Signal., 26: 383-97 (2014) 2014
3',5'-cyclic nucleotide phosphodiesterases (PDEs) are the only known enzymes to compartmentalize cAMP and cGMP, yet little is known about how PDEs are dynamically regulated across the lifespan. We mapped mRNA expression of all 21 PDE isoforms in the adult rat and mouse central nervous system (CNS) using quantitative polymerase chain reaction (qPCR) and in situ hybridization to assess conservation across species. We also compared PDE mRNA and protein in the brains of old (26 months) versus young (5 months) Sprague-Dawley rats, with select experiments replicated in old (9 months) versus young (2 months) BALB/cJ mice. We show that each PDE isoform exhibits a unique expression pattern across the brain that is highly conserved between rats, mice, and humans. PDE1B, PDE1C, PDE2A, PDE4A, PDE4D, PDE5A, PDE7A, PDE8A, PDE8B, PDE10A, and PDE11A showed an age-related increase or decrease in mRNA expression in at least 1 of the 4 brain regions examined (hippocampus, cortex, striatum, and cerebellum). In contrast, mRNA expression of PDE1A, PDE3A, PDE3B, PDE4B, PDE7A, PDE7B, and PDE9A did not change with age. Age-related increases in PDE11A4, PDE8A3, PDE8A4/5, and PDE1C1 protein expression were confirmed in hippocampus of old versus young rodents, as were age-related increases in PDE8A3 protein expression in the striatum. Age-related changes in PDE expression appear to have functional consequences as, relative to young rats, the hippocampi of old rats demonstrated strikingly decreased phosphorylation of GluR1, CaMKIIα, and CaMKIIβ, decreased expression of the transmembrane AMPA regulatory proteins γ2 (a.k.a. stargazin) and γ8, and increased trimethylation of H3K27. Interestingly, expression of PDE11A4, PDE8A4/5, PDE8A3, and PDE1C1 correlate with these functional endpoints in young but not old rats, suggesting that aging is not only associated with a change in PDE expression but also a change in PDE compartmentalization.
|Postsynaptic density scaffold SAP102 regulates cortical synapse development through EphB and PAK signaling pathway. |
Murata, Yasunobu and Constantine-Paton, Martha
J. Neurosci., 33: 5040-52 (2013) 2013
Membrane-associated guanylate kinases (MAGUKs), including SAP102, PSD-95, PSD-93, and SAP97, are scaffolding proteins for ionotropic glutamate receptors at excitatory synapses. MAGUKs play critical roles in synaptic plasticity; however, details of signaling roles for each MAGUK remain largely unknown. Here we report that SAP102 regulates cortical synapse development through the EphB and PAK signaling pathways. Using lentivirus-delivered shRNAs, we found that SAP102 and PSD-95, but not PSD-93, are necessary for excitatory synapse formation and synaptic AMPA receptor (AMPAR) localization in developing mouse cortical neurons. SAP102 knockdown (KD) increased numbers of elongated dendritic filopodia, which is often observed in mouse models and human patients with mental retardation. Further analysis revealed that SAP102 coimmunoprecipitated the receptor tyrosine kinase EphB2 and RacGEF Kalirin-7 in neonatal cortex, and SAP102 KD reduced surface expression and dendritic localization of EphB. Moreover, SAP102 KD prevented reorganization of actin filaments, synapse formation, and synaptic AMPAR trafficking in response to EphB activation triggered by its ligand ephrinB. Last, p21-activated kinases (PAKs) were downregulated in SAP102 KD neurons. These results demonstrate that SAP102 has unique roles in cortical synapse development by mediating EphB and its downstream PAK signaling pathway. Both SAP102 and PAKs are associated with X-linked mental retardation in humans; thus, synapse formation mediated by EphB/SAP102/PAK signaling in the early postnatal brain may be crucial for cognitive development.
|TNF-α Downregulates Inhibitory Neurotransmission through Protein Phosphatase 1-Dependent Trafficking of GABAA Receptors. |
Pribiag, Horia and Stellwagen, David
J. Neurosci., 33: 15879-93 (2013) 2013
Inflammation has been implicated in the progression of neurological disease, yet precisely how inflammation affects neuronal function remains unclear. Tumor necrosis factor-α (TNFα) is a proinflammatory cytokine that regulates synapse function by controlling neurotransmitter receptor trafficking and homeostatic synaptic plasticity. Here we characterize the mechanisms through which TNFα regulates inhibitory synapse function in mature rat and mouse hippocampal neurons. Acute application of TNFα induces a rapid and persistent decrease of inhibitory synaptic strength and downregulation of cell-surface levels of GABAARs containing α1, α2, β2/3, and γ2 subunits. We show that trafficking of GABAARs in response to TNFα is mediated by neuronally expressed TNF receptor 1 and requires activation of p38 MAPK, phosphatidylinositol 3-kinase, protein phosphatase 1 (PP1), and dynamin GTPase. Furthermore, TNFα enhances the association of PP1 with GABAAR β3 subunits and dephosphorylates a site on β3 known to regulate phospho-dependent interactions with the endocytic machinery. Conversely, we find that calcineurin and PP2A are not essential components of the signaling pathway and that clustering of the scaffolding protein gephyrin is only reduced after the initial receptor endocytosis. Together, these findings demonstrate a distinct mechanism of regulated GABAAR endocytosis that may contribute to the disruption of circuit homeostasis under neuroinflammatory conditions.
|Selective loss of AMPA receptors at corticothalamic synapses in the epileptic stargazer mouse. |
Z Barad,O Shevtsova,G W Arbuthnott,B Leitch
Neuroscience 217 2012
Absence seizures are common in the stargazer mutant mouse. The mutation underlying the epileptic phenotype in stargazers is a defect in the gene encoding the normal expression of the protein stargazin. Stargazin is involved in the membrane trafficking and synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at excitatory glutamatergic synapses. Thus, the genetic defect in the stargazer results in a loss of AMPARs and consequently, excitation at glutamatergic synapses. Absence seizures are known to arise in thalamocortical networks. In the present study we show for the first time, using Western blot analysis and quantitative immunogold cytochemistry, that in the epileptic stargazer mouse, there is a global loss of AMPAR protein in nucleus reticularis (RTN) and a selective loss of AMPARs at corticothalamic synapses in inhibitory neurons of the RTN thalamus. In contrast, there is no significant loss of AMPARs at corticothalamic synapses in excitatory relay neurons in the thalamic ventral posterior (VP) region. The findings of this study thus provide cellular and molecular evidence for a selective regional loss of synaptic AMPAR within the RTN that could account for the loss of function at these inhibitory neuron synapses, which has previously been reported from electrophysiological studies. The specific loss of AMPARs at RTN but not relay synapses in the thalamus of the stargazer, could contribute to the absence epilepsy phenotype by altering thalamocortical network oscillations. This is supported by recent evidence that loss of glutamate receptor subunit 4 (GluA4) (the predominant AMPAR-subtype in the thalamus), also leads to a specific reduction in strength in the cortico-RTN pathway and enhanced thalamocortical oscillations, in the Gria4(-/-) model of absence epilepsy. Thus further study of thalamic changes in these models could be important for future development of drugs targeted to absence epilepsy.
|Genetic Deletion of NR3A Accelerates Glutamatergic Synapse Maturation. |
Maile A Henson,Rylan S Larsen,Shelikha N Lawson,Isabel Pérez-Otaño,Nobuki Nakanishi,Stuart A Lipton,Benjamin D Philpot
PloS one 7 2012
Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.
|Altered glutamate receptor function in the cerebellum of the Ppt1-/- mouse, a murine model of infantile neuronal ceroid lipofuscinosis. |
Rozzy Finn,Attila D Kovács,David A Pearce
Journal of neuroscience research 90 2012
The neuronal ceroid lipofuscinoses (NCLs) are a family of devastating pediatric neurodegenerative disorders and currently represent the most common form of pediatric-onset neurodegeneration. Infantile NCL (INCL), the most aggressive of these disorders, is caused by mutations in the CLN1 gene that encodes the enzyme palmitoyl protein thioesterase 1 (PPT1). Previous studies have suggested that glutamatergic neurotransmission may be disrupted in INCL, so the present study investigates glutamate receptor function in the Ppt1(-/-) mouse model of INCL by comparing the sensitivity of cultured wild-type (WT) and Ppt1(-/-) cerebellar granule cells to glutamate receptor-mediated toxicity. Ppt1(-/-) neurons were significantly less sensitive to AMPA receptor-mediated toxicity but markedly more vulnerable to NMDA receptor-mediated cell death. Because glutamate receptor function is regulated primarily by the surface expression level of the receptor, the surface level of AMPA and NMDA receptor subunits in the cerebella of WT and Ppt1(-/-) mice was also examined. Western blotting of surface cross-linked cerebellar samples showed a significantly lower surface level of the GluR4 AMPA receptor subunit in Ppt1(-/-) mice, providing a plausible explanation for the decreased vulnerability of Ppt1(-/-) cerebellar neurons to AMPA receptor-mediated cell death. The surface expression of the NR1, NR2A, and NR2B NMDA receptor subunits was similar in the cerebella of WT and Ppt1(-/-) mice, indicating that there is another mechanism behind the increased sensitivity of Ppt1(-/-) cerebellar granule cells to NMDA toxicity. Our results indicate an AMPA receptor hypofunction and NMDA receptor hyperfunction phenotype in Ppt1(-/-) neurons and provide new therapeutic targets for INCL.
|Deficits in LTP Induction by 5-HT2A Receptor Antagonist in a Mouse Model for Fragile X Syndrome. |
Zhao-Hui Xu,Qi Yang,Lan Ma,Shui-Bing Liu,Guang-Sheng Chen,Yu-Mei Wu,Xiao-Qiang Li,Gang Liu,Ming-Gao Zhao
PloS one 7 2012
Fragile X syndrome is a common inherited form of mental retardation caused by the lack of fragile X mental retardation protein (FMRP) because of Fmr1 gene silencing. Serotonin (5-HT) is significantly increased in the null mutants of Drosophila Fmr1, and elevated 5-HT brain levels result in cognitive and behavioral deficits in human patients. The serotonin type 2A receptor (5-HT2AR) is highly expressed in the cerebral cortex; it acts on pyramidal cells and GABAergic interneurons to modulate cortical functions. 5-HT2AR and FMRP both regulate synaptic plasticity. Therefore, the lack of FMRP may affect serotoninergic activity. In this study, we determined the involvement of FMRP in the 5-HT modulation of synaptic potentiation with the use of primary cortical neuron culture and brain slice recording. Pharmacological inhibition of 5-HT2AR by R-96544 or ketanserin facilitated long-term potentiation (LTP) in the anterior cingulate cortex (ACC) of WT mice. The prefrontal LTP induction was dependent on the activation of NMDARs and elevation of postsynaptic Ca(2+) concentrations. By contrast, inhibition of 5-HT2AR could not restore the induction of LTP in the ACC of Fmr1 knock-out mice. Furthermore, 5-HT2AR inhibition induced AMPA receptor GluR1 subtype surface insertion in the cultured ACC neurons of Fmr1 WT mice, however, GluR1 surface insertion by inhibition of 5-HT2AR was impaired in the neurons of Fmr1KO mice. These findings suggested that FMRP was involved in serotonin receptor signaling and contributed in GluR1 surface expression induced by 5-HT2AR inactivation.
|Rapid enrichment of presynaptic protein in boutons undergoing classical conditioning is mediated by brain-derived neurotrophic factor. |
Li, W and Keifer, J
Neuroscience, 203: 50-8 (2012) 2012
Presynaptic structural modifications are thought to accompany activity-dependent synaptic plasticity and learning. This may involve the conversion of nonfunctional synapses into active ones or the generation of entirely new synapses. Here, using an in vitro neural analog of classical conditioning, we investigated presynaptic structural changes restricted to auditory nerve synapses that convey the conditioned stimulus (CS) by tract tracing using fluorescent tracers combined with immunostaining for the synaptic vesicle-associated protein synaptophysin. The results show that the size of presynaptic auditory boutons increased and the area and fluorescence intensity of punctate staining for synaptophysin were enhanced after conditioning. This occurred only for auditory nerve boutons apposed to the dendrites but not the somata of abducens motor neurons. Conditioning increased the percentage of boutons that were immunopositive for synaptophysin and enhanced the number of synaptophysin puncta they contained. Pretreatment with antibodies against brain-derived neurotrophic factor (BDNF) inhibited these conditioning-induced structural changes. There was also a net increase in the number of boutons apposed to abducens motor neurons after conditioning or BDNF treatment. These data indicate that the rapid enrichment of presynaptic boutons with proteins required for neurotransmitter recycling and release occurs during classical conditioning and that these processes are mediated by BDNF.
|Distinct regional and subcellular localization of the actin-binding protein filamin a in the mature rat brain. |
Yoav Noam,Lise Phan,Shawn McClelland,Erik M Manders,Markus U Ehrengruber,Wytse J Wadman,Tallie Z Baram,Yuncai Chen
The Journal of comparative neurology 520 2012
Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain. Here we characterize in detail the neuroanatomical distribution and subcellular localization of FLNa in the mature rat brain, by using two antisera directed against epitopes at either the N' or the C' terminus of the protein, further validated by mRNA expression. FLNa was widely and selectively expressed throughout the brain, and the intensity of immunoreactivity was region dependent. The most intensely FLNa-labeled neurons were found in discrete neuronal systems, including basal forebrain structures, anterior nuclear group of thalamus, and hypothalamic parvocellular neurons. Pyramidal neurons in neocortex and hippocampus and magnocellular cells in basolateral amygdaloid nucleus were also intensely FLNa immunoreactive, and strong FLNa labeling was evident in the pontine and medullary raphe nuclei and in sensory and spinal trigeminal nuclei. The subcellular localization of FLNa was evaluated in situ as well as in primary hippocampal neurons. Punctate expression was found in somata and along the dendritic shaft, but FLNa was not detected in dendritic spines. These subcellular distribution patterns were recapitulated in hippocampal and neocortical pyramidal neurons in vivo. The characterization of the expression and subcellular localization of FLNa may provide new clues to the functional roles of this cytoskeletal protein in the adult brain. J. Comp. Neurol. 520:3013-3034, 2012. © 2012 Wiley Periodicals, Inc.
|Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant. |
Zheng, Zhaoqing, et al.
J. Neurophysiol., 108: 101-11 (2012) 2012
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.
|Effects of acute cocaine or dopamine receptor agonists on AMPA receptor distribution in the rat nucleus accumbens. |
Ferrario CR, Li X, Wolf ME.
Synapse (New York, N.Y.) 65 54-63 2011
Changes in α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) surface expression in the rodent nucleus accumbens (NAc) are produced by cocaine exposure and implicated in addiction-related behaviors. The direction of change depends on the animal's prior drug history. However, little is known about the effect of a single exposure to cocaine on AMPAR distribution in the NAc of untreated rats. This is essential information for interpreting the literature on AMPAR trafficking after repeated cocaine exposure. In this study, we used a protein crosslinking assay to determine the effect of a single cocaine injection on surface and intracellular AMPAR subunit levels in the rat NAc. We found increased AMPAR surface expression in the NAc 24 h, but not 30 min or 2 h, after cocaine injection. A major effect of cocaine is to increase extracellular dopamine (DA) levels, leading to DA receptor activation. Therefore, we also evaluated the effects of directly acting DA receptor agonists. In contrast to the effects of cocaine, AMPAR surface expression was significantly decreased 24 h after injection of the D2-class agonist quinpirole, whereas no significant effects were produced by the D1-class agonist SKF 81297 or the mixed DA agonist apomorphine. Our results show that the effects of a single cocaine exposure in drug- and injection-naïve rats are distinct from those previously reported after repeated cocaine administration. They further suggest that cocaine exerts these effects by influencing neuronal circuits rather than simply stimulating NAc DA transmission.
|Immunohistochemical identification and synaptic inputs to the diffuse bipolar cell type DB1 in macaque retina. |
Puthussery T, Gayet-Primo J, Taylor WR, Haverkamp S.
The Journal of comparative neurology 519 3640-56 2011
Detailed analysis of the synaptic inputs to the primate DB1 bipolar cell has been precluded by the absence of a suitable immunohistochemical marker. Here we demonstrate that antibodies for the EF-hand calcium-binding protein, secretagogin, strongly label the DB1 bipolar cell as well as a mixed population of GABAergic amacrine cells in the macaque retina. Using secretagogin as a marker, we show that the DB1 bipolar makes synaptic contact with both L/M as well as S-cone photoreceptors and only minimal contact with rod photoreceptors. Electron microscopy showed that the DB1 bipolar makes flat contacts at both triad-associated and nontriad-associated positions on the cone pedicle. Double labeling with various glutamate receptor subunit antibodies failed to conclusively determine the subunit composition of the glutamate receptors on DB1 bipolar cells. In the IPL, DB1 bipolar cell axon terminals expressed the glycine receptor, GlyRα1, at sites of contact with AII amacrine cells, suggesting that these cells receive input from the rod pathway. J. Comp. Neurol. 519:3640-3656, 2011. © 2011 Wiley-Liss, Inc.
|Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca(2+)-permeable AMPA receptors during the incubation of cocaine craving. |
Ferrario CR, Loweth JA, Milovanovic M, Ford KA, Galiñanes GL, Heng LJ, Tseng KY, Wolf ME
Cue-induced cocaine seeking intensifies or incubates after withdrawal from extended access cocaine self-administration, a phenomenon termed incubation of cocaine craving. The expression of incubated craving is mediated by Ca(2+)-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). Thus, CP-AMPARs are a potential target for therapeutic intervention, making it important to understand mechanisms that govern their accumulation. Here we used subcellular fractionation and biotinylation of NAc tissue to examine the abundance and distribution of AMPAR subunits, and GluA1 phosphorylation, in the incubation model. We also studied two transmembrane AMPA receptor regulatory proteins (TARPs), γ-2 and γ-4. Our results, together with earlier findings, suggest that some of the new CP-AMPARs are synaptic. These are probably associated with γ-2, but they are loosely tethered to the PSD. Levels of GluA1 phosphorylated at serine 845 (pS845 GluA1) were significantly increased in biotinylated tissue and in an extrasynaptic membrane-enriched fraction. These results suggest that increased synaptic levels of CP-AMPARs may result in part from an increase in pS845 GluA1 in extrasynaptic membranes, given that S845 phosphorylation primes GluA1-containing AMPARs for synaptic insertion and extrasynaptic AMPARs supply the synapse. Some of the new extrasynaptic CP-AMPARs are likely associated with γ-4, rather than γ-2. The maintenance of CP-AMPARs in NAc synapses during withdrawal is accompanied by activation of CaMKII and ERK2 but not CaMKI. Overall, AMPAR plasticity in the incubation model shares some features with better described forms of synaptic plasticity, although the timing of the phenomenon and the persistence of related neuroadaptations are significantly different.Copyright © 2011 Elsevier Ltd. All rights reserved.
|Acute neuregulin-1 signaling influences AMPA receptor mediated responses in cultured cerebellar granule neurons. |
Fenster C, Vullhorst D, Buonanno A
Brain research bulletin 2011
Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10-12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of high-glyine/0 Mg(2+) (which activates synaptic NMDARs) on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar to Schaeffer collateral to CA1 synapses, NRG1 effects are activity dependent and mediated via modulation of synaptic AMPARs.Copyright © 2011 Elsevier Inc. All rights reserved.
|Cannabinoid receptor type 1 expression during postnatal development of the rat retina. |
Zabouri N, Bouchard JF, Casanova C
J Comp Neurol 519 1258-80. doi 2011
|Cornichon-2 Modulates aMPA receptor-transmembrane aMPA receptor regulatory protein assembly to dictate gating and pharmacology. |
Gill MB, Kato AS, Roberts MF, Yu H, Wang H, Tomita S, Bredt DS
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 6928-38. 2011
Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon-2/3 (CNIH-2/3). The mechanisms that control AMPA receptor complex assembly remain unclear. AMPA receptor responses in neurons differ from those in cell lines transfected with GluA plus TARPs γ-8 or γ-7, which show unusual resensitization kinetics and non-native AMPA receptor pharmacologies. Using tandem GluA/TARP constructs to constrain stoichiometry, we show here that these peculiar kinetic and pharmacological signatures occur in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuron-like kinetics and pharmacologies, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that coexpression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface in a γ-8-dependent manner to dictate receptor pharmacology. In the cerebellum, however, CNIH-2 expressed in Purkinje neurons does not reach the neuronal surface. In concordance, stargazer Purkinje neurons, which express CNIH-2 and γ-7, display AMPA receptor kinetics/pharmacologies that can only be recapitulated recombinantly by a low γ-7/GluA stoichiometry. Together, these data suggest that CNIH-2 modulates neuronal AMPA receptor auxiliary subunit assembly by regulating the number of TARPs within an AMPA receptor complex to modulate receptor gating and pharmacology.
|Gestational nicotine exposure regulates expression of aMPA and nMDA receptors and their signaling apparatus in developing and adult rat hippocampus. |
Wang H, Dávila-García MI, Yarl W, Gondré-Lewis MC
Neuroscience 188 168-81. Epub 2011 May 12. 2011
Untimely activation of nicotinic acetylcholine receptors (nAChRs) by nicotine results in short- and long-term consequences on learning and behavior. In this study, the aim was to determine how prenatal nicotine exposure affects components of glutamatergic signaling in the hippocampus during postnatal development. We investigated regulation of both nAChRs and glutamate receptors for AMPA and N-methyl-d-aspartate (NMDA), from postnatal day 1 (P1) to P63 after a temporally restricted exposure to saline or nicotine for 14 days in utero. We analyzed postsynaptic density components associated with AMPA receptor (AMPAR) and NMDA receptor (NMDAR) signaling: calmodulin (CaM), CaM Kinase II alpha (CaMKIIα), and postsynaptic density-95 (PSD95), as well as presynaptically localized synaptosomal-associated protein 25 (SNAP25). At P1, there was significantly heightened expression of AMPAR subunit GluR1 but not GluR2, and of NMDAR subunits NR1, NR2a, and NR2d but not NR2b. NR2c was not detectable. CaM, CaMKIIα, and PSD95 were also significantly upregulated at P1, together with presynaptic SNAP25. This enhanced expression of glutamate receptors and signaling proteins was concomitant with elevated levels of [(3)H]epibatidine ([(3)H]EB) binding in prenatal nicotine-exposed hippocampus, indicating that α4β2 nAChR may influence glutamatergic function in the hippocampus at P1. By P14, neither [(3)H]EB binding nor the expression levels of subunits GluR1, GluR2, NR1, NR2a, NR2b, NR2c, or NR2d seemed changed with prenatal nicotine. However, CaMKIIα was significantly upregulated with nicotine treatment while CaM showed downregulation at P14. The effects of nicotine persisted in P63 young adult brains which exhibited significantly downregulated GluR2, NR1, and NR2c expression levels in hippocampal homogenates and a considerably muted overall distribution of [(3)H]AMPA binding in areas CA1, CA2 and CA3, and the dentate gyrus. Our results suggest that prenatal nicotine exposure can regulate the glutamatergic signaling system throughout postnatal development by enhancing or inhibiting availability of AMPAR and NMDAR or their signaling components. The persistent depression, in adults, of the requisite NR1 subunit for NMDAR assembly, and of GluR2, important for assembly, trafficking, and biophysical properties of AMPAR, indicates that nicotine may alter ionotropic glutamate receptor stoichiometry and functional properties in adults after prenatally restricted nicotine exposure.Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
|Phosphodiesterase 11A in brain is enriched in ventral hippocampus and deletion causes psychiatric disease-related phenotypes. |
Kelly MP, Logue SF, Brennan J, Day JP, Lakkaraju S, Jiang L, Zhong X, Tam M, Sukoff Rizzo SJ, Platt BJ, Dwyer JM, Neal S, Pulito VL, Agostino MJ, Grauer SM, Navarra RL, Kelley C, Comery TA, Murrills RJ, Houslay MD, Brandon NJ.
Proc Natl Acad Sci U S A 2010
Phosphodiesterase 11A (PDE11A) is the most recently identified family of phosphodiesterases (PDEs), the only known enzymes to break down cyclic nucleotides. The tissue expression profile of this dual specificity PDE is controversial, and little is understood of its biological function, particularly in the brain. We seek here to determine if PDE11A is expressed in the brain and to understand its function, using PDE11A(-/-) knockout (KO) mice. We show that PDE11A mRNA and protein are largely restricted to hippocampus CA1, subiculum, and the amygdalohippocampal area, with a two- to threefold enrichment in the ventral vs. dorsal hippocampus, equal distribution between cytosolic and membrane fractions, and increasing levels of protein expression from postnatal day 7 through adulthood. Interestingly, PDE11A KO mice show subtle psychiatric-disease-related deficits, including hyperactivity in an open field, increased sensitivity to the glutamate N-methyl-D-aspartate receptor antagonist MK-801, as well as deficits in social behaviors (social odor recognition memory and social avoidance). In addition, PDE11A KO mice show enlarged lateral ventricles and increased activity in CA1 (as per increased Arc mRNA), phenotypes associated with psychiatric disease. The increased sensitivity to MK-801 exhibited by PDE11A KO mice may be explained by the biochemical dysregulation observed around the glutamate alpha-amino-3-hydroxy-5-methyl-4-isozazolepropionic (AMPA) receptor, including decreased levels of phosphorylated-GluR1 at Ser845 and the prototypical transmembrane AMPA-receptor-associated proteins stargazin (gamma2) and gamma8. Together, our data provide convincing evidence that PDE11A expression is restricted in the brain but plays a significant role in regulating brain function.
|Increased insertion of GluR2-lacking AMPA receptors at hippocampal synapses upon repeated morphine administration. |
Billa SK, Liu J, Bjorklund NL, Sinha N, Fu Y, Shinnick-Gallagher P, MorÃ³n JA
Molecular pharmacology 77 874-83 Epub 2010 Feb 16 2010
Evidence suggests that the long-term adaptations in the hippocampus after repeated drug treatment may parallel its role during memory formation. The neuroplasticity that subserves learning and memory is also believed to underlie addictive processes. We have reported previously that repeated morphine administration alters local distribution of endocytic proteins at hippocampal synapses, which could in turn affect expression of glutamate receptors. Glutamatergic systems, including alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), are believed to be involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are only beginning to be understood. The present study further examines the effects of repeated morphine administration on the expression and composition of AMPARs and the functional ramifications. Twelve hours after the last morphine injection, we observed an increased expression of AMPARs lacking glutamate receptor (GluR) 2 in hippocampal synaptic fractions. Immunoblotting studies show that 12 h after morphine treatment, GluR1 subunits are increased at the postsynaptic density (PSD) and at extrasynaptic sites, whereas GluR3 subunits are only increased at the PSD, and they show how this alters receptor subunit composition. In addition, we provide electrophysiological evidence that AMPARs are switched to Ca(2+)-permeable (GluR2-lacking) at the synapse 12 h after repeated morphine treatment, affecting the magnitude of long-term depression at hippocampal neurons. We propose that morphine-induced changes in glutamatergic synaptic transmission in the hippocampus may play an important role in the neuroadaptations induced by repeated morphine administration.
|Morphological Changes and Synaptogenesis of Corticothalamic Neurons in the Somatosensory Cortex of Rat during Perinatal Development. |
Hsu CI, Ho TS, Liou YR, Chang YC
Cereb Cortex 2010
When rat fetuses grew from embryonic day (E) 18 to the day of birth (P0), the corticothalamic (CT) neurons, as identified by back labeling with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI), in the somatosensory cortex underwent gradual changes in the shape of their cell bodies, in their distribution in the cortical plate and in the complexity of dendritic branching. Fluorescence immunocytochemical studies indicated that in the marginal zone (MZ) the apical dendrites of the CT neurons formed contacts with horizontally oriented axons and contained putative glutamatergic, as clusters exhibiting both synaptophysin and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit immunoreactivities, and gamma-aminobutyric acid (GABA)-ergic synapses, as clusters exhibiting both synaptophysin and gephyrin immunoreactivities. Quantitative analyses further revealed that during this perinatal period, the proportion of CT neurons containing glutamatergic synapses increased significantly, whereas the proportion of CT neurons containing GABAergic synapses remained virtually unchanged. Our results indicate that glutamatergic and GABAergic synapses between the CT neurons and the axons in the MZ are already formed in rat cortices as early as E18 and further suggest that the activities of the neural networks in the somatosensory cortex could be conveyed to their targets in the thalamus in rat brains at least 3 days before birth.
|Quantitative analysis of AMPA receptor subunit composition in addiction-related brain regions. |
Reimers JM, Milovanovic M, Wolf ME
Brain Res 2010
The subunit composition of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) is an important determinant of AMPAR biophysical properties and trafficking. To date, AMPAR subunit composition has been quantitatively evaluated only for the hippocampus, where different experimental approaches have yielded different results. Here, we used quantitative co-immunoprecipitation to characterize GluA1-3 associations in the adult rat nucleus accumbens, dorsal striatum, prefrontal cortex, and hippocampus, and blue native electrophoresis (BNE) to study GluA1-3 assembly state. In all brain regions, co-immunoprecipitation experiments showed that ~90% of GluA1 was associated with GluA2 or GluA3 (most was GluA1A2). All regions contained a small number of GluA1A3 receptors. Homomeric GluA1 receptors may also exist. More than half of the GluA2 (53%-65% depending on the region) was not associated with GluA1. However, this represents an over-estimate of the percent of GluA2 present in GluA2A3 receptors, based on BNE results demonstrating that the majority of GluA2 exists as dimers, rather than functional tetrameric receptors. Relatively more GluA1 was present in tetramers. Together with other findings, our results suggest a dominant role for GluA1A2 receptors in all brain regions examined. They also help explain why different results for hippocampal AMPAR subunit composition were obtained using co-immunoprecipitation, which assesses the total cellular pool of AMPARs including partially assembled AMPARs in intracellular compartments, and electrophysiological approaches, which can selectively assess tetrameric (functional) AMPARs on the cell surface.
|Morphine Induces AMPA Receptor Internalization in Primary Hippocampal Neurons via Calcineurin-Dependent Dephosphorylation of GluR1 Subunits. |
Kam AY, Liao D, Loh HH, Law PY
J Neurosci 30 15304-16. 2010
Chronic morphine treatment resulting in the alteration of postsynaptic levels of AMPA receptors, thereby modulating synaptic strength, has been reported. However, the mechanism underlying such drug-induced synaptic modification has not been resolved. By monitoring the GluR1 trafficking in primary hippocampal neurons using the pHluorin-GluR1 imaging and biotinylation studies, we observed that prolonged morphine exposure significantly induced loss of synaptic and extrasynaptic GluR1 by internalization. The morphine-induced GluR1 endocytosis was independent of neural network activities or NMDA receptor activities, as neither blocking the sodium channels with tetrodotoxin nor NMDA receptors with dl-APV altered the effects of morphine. Instead, morphine-induced GluR1 endocytosis is attributed to a change in the phosphorylation state of the GluR1 at Ser(845) as morphine significantly decreased the dephosphorylation of GluR1 at this site. Such changes in Ser(845) phosphorylation required morphine-induced activation of calcineurin, based on the observations that a calcineurin inhibitor, FK506, completely abrogated the dephosphorylation, and morphine treatment led to an increase in calcineurin enzymatic activity, even in the presence of dl-APV. Importantly, pretreatment with FK506 and overexpression of the GluR1 mutants, S845D (phospho-mimic) or S845A (phospho-blocking) attenuated the morphine-induced GluR1 endocytosis. Therefore, the calcineurin-mediated GluR1-S845 dephosphorylation is critical for the morphine-induced changes in the postsynaptic AMPA receptor level. Together, these findings reveal a novel molecular mechanism for opioid-induced neuronal adaptation and/or synaptic impairment.
|Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate. |
Mayumi Sugimoto,Shinji Sasaki,Toshio Watanabe,Shota Nishimura,Atsushi Ideta,Maya Yamazaki,Keiko Matsuda,Michisuke Yuzaki,Kenji Sakimura,Yoshito Aoyagi,Yoshikazu Sugimoto
"PloS one" 5 2010
Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.Full Text Article
|Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication. |
Ozlem Bozdagi,Takeshi Sakurai,Danae Papapetrou,Xiaobin Wang,Dara L Dickstein,Nagahide Takahashi,Yuji Kajiwara,Mu Yang,Adam M Katz,Maria Luisa Scattoni,Mark J Harris,Roheeni Saxena,Jill L Silverman,Jacqueline N Crawley,Qiang Zhou,Patrick R Hof,Joseph D Buxbaum
Molecular autism 1 2010
SHANK3 is a protein in the core of the postsynaptic density (PSD) and has a critical role in recruiting many key functional elements to the PSD and to the synapse, including components of ?-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA), metabotropic glutamate (mGlu) and N-methyl-D-aspartic acid (NMDA) glutamate receptors, as well as cytoskeletal elements. Loss of a functional copy of the SHANK3 gene leads to the neurobehavioral manifestations of 22q13 deletion syndrome and/or to autism spectrum disorders. The goal of this study was to examine the effects of haploinsufficiency of full-length Shank3 in mice, focusing on synaptic development, transmission and plasticity, as well as on social behaviors, as a model for understanding SHANK3 haploinsufficiency in humans.Full Text Article
|Altered sensitivity to excitotoxic cell death and glutamate receptor expression between two commonly studied mouse strains. |
Rozzy Finn,Attila D Kovács,David A Pearce
Journal of neuroscience research 88 2010
Alterations in glutamatergic synapse function have been implicated in the pathogenesis of many different neurological disorders, including ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, and Huntington's disease. While studying glutamate receptor function in juvenile Batten disease on the C57BL/6J and 129S6/S(v)E(v) mouse backgrounds, we noticed differences unlikely to be due to mutation difference alone. We report here that primary cerebellar granule cell cultures from C57BL/6J mice are more sensitive to N-methyl-D-aspartate (NMDA)-mediated cell death. Moreover, sensitivity to AMPA-mediated excitotoxicity is more variable and is dependent on the treatment conditions and age of the cultures. Glutamate receptor surface expression levels examined in vitro by in situ ELISA and in vivo by Western blot in surface cross-linked cerebellar samples indicated that these differences in sensitivity likely are due to strain-dependent differences in cell surface receptor expression levels. We propose that differences in glutamate receptor expression and in excitotoxic vulnerability should be taken into consideration in the context of characterizing disease models on the C57BL/6J and 129S6/S(v)E(v) mouse backgrounds.Full Text Article
|Altered neurotransmission in the mesolimbic reward system of Girk mice. |
Arora D, Haluk DM, Kourrich S, Pravetoni M, Fernández-Alacid L, Nicolau JC, Luján R, Wickman K
J Neurochem 114 1487-97. Epub 2010 Jun 16. 2010
Mice lacking the Girk2 subunit of G protein-gated inwardly rectifying K+ (Girk) channels exhibit dopamine-dependent hyperactivity and elevated responses to drugs that stimulate dopamine neurotransmission. The dopamine-dependent phenotypes seen in Girk2(-/-) mice could reflect increased intrinsic excitability of or diminished inhibitory feedback to midbrain dopamine neurons, or secondary adaptations triggered by Girk2 ablation. We addressed these possibilities by evaluating Girk(-/-) mice in behavioral, electrophysiological, and cell biological assays centered on the mesolimbic dopamine system. Despite differences in the contribution of Girk1 and Girk2 subunits to Girk signaling in midbrain dopamine neurons, Girk1(-/-) and Girk2(-/-) mice exhibited comparable baseline hyperactivities and enhanced responses to cocaine. Girk ablation also correlated with altered afferent input to dopamine neurons in the ventral tegmental area. Dopamine neurons from Girk1(-/-) and Girk2(-/-) mice exhibited elevated glutamatergic neurotransmission, paralleled by increased synaptic levels of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate glutamate receptors. In addition, synapse density, alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor levels, and glutamatergic neurotransmission were elevated in medium spiny neurons of the nucleus accumbens from Girk1(-/-) and Girk2(-/-) mice. We conclude that dopamine-dependent phenotypes in Girk2(-/-) mice are not solely attributable to a loss of Girk signaling in dopamine neurons, and likely involve secondary adaptations facilitating glutamatergic signaling in the mesolimbic reward system.Full Text Article
|Excitatory-inhibitory relationship in the fascia dentata in the Ts65Dn mouse model of Down syndrome. |
Pavel V Belichenko,Alexander M Kleschevnikov,Eliezer Masliah,Chengbiao Wu,Ryoko Takimoto-Kimura,Ahmad Salehi,William C Mobley
The Journal of comparative neurology 512 2009
Down syndrome (DS) is a neurological disorder causing impaired learning and memory. Partial trisomy 16 mice (Ts65Dn) are a genetic model for DS. Previously, we demonstrated widespread alterations of pre- and postsynaptic elements and physiological abnormalities in Ts65Dn mice. The average diameter of presynaptic boutons and spines in the neocortex and hippocampus was enlarged. Failed induction of long-term potentiation (LTP) due to excessive inhibition was observed. In this paper we investigate the morphological substrate for excessive inhibition in Ts65Dn. We used electron microscopy (EM) to characterize synapses, confocal microscopy to analyze colocalization of the general marker for synaptic vesicle protein with specific protein markers for inhibitory and excitatory synapses, and densitometry to characterize the distribution of the receptor and several proteins essential for synaptic clustering of neurotransmitter receptors. EM analysis of synapses in the Ts65Dn vs. 2N showed that synaptic opposition lengths were significantly greater for symmetric synapses (approximately 18%), but not for asymmetric ones. Overall, a significant increase in colocalization coefficients of glutamic acid decarboxylase (GAD)65/p38 immunoreactivity (IR) (approximately 27%) and vesicular GABA transporter (VGAT)/p38 IR (approximately 41%) was found, but not in vesicular glutamate transporter 1 (VGLUT1)/p38 IR. A significant overall decrease of IR in the hippocampus of Ts65Dn mice compared with 2N mice for glutamate receptor 2 (GluR2; approximately 13%) and anti-gamma-aminobutyric acid (GABA)(A) receptor beta2/3 subunit (approximately 20%) was also found. The study of proteins essential for synaptic clustering of receptors revealed a significant increase in puncta size for neuroligin 2 (approximately 13%) and GABA(A) receptor-associated protein (GABARAP; approximately 13%), but not for neuroligin 1 and gephyrin. The results demonstrate a significant alteration of inhibitory synapses in the fascia dentata of Ts65Dn mice.
|Ampakines cause sustained increases in brain-derived neurotrophic factor signaling at excitatory synapses without changes in AMPA receptor subunit expression. |
J C Lauterborn, E Pineda, L Y Chen, E A Ramirez, G Lynch, C M Gall, J C Lauterborn, E Pineda, L Y Chen, E A Ramirez, G Lynch, C M Gall, J C Lauterborn, E Pineda, L Y Chen, E A Ramirez, G Lynch, C M Gall, J C Lauterborn, E Pineda, L Y Chen, E A Ramirez, G Lynch, C M Gall
Neuroscience 159 283-95 2009
Recent demonstrations that positive modulators of AMPA-type glutamate receptors (ampakines) increase neuronal brain-derived neurotrophic factor (BDNF) expression have suggested a novel strategy for treating neurodegenerative diseases. However, reports that AMPA and BDNF receptors are down-regulated by prolonged activation raise concerns about the extent to which activity-induced increases in BDNF levels can be sustained without compromising glutamate receptor function. The present study constitutes an initial test of whether ampakines can cause enduring increases in BDNF content and signaling without affecting AMPA receptor (AMPAR) expression. Prolonged (12-24 h) treatment with the ampakine CX614 reduced AMPAR subunit (glutamate receptor subunit (GluR) 1-3) mRNA and protein levels in cultured rat hippocampal slices whereas treatment with AMPAR antagonists had the opposite effects. The cholinergic agonist carbachol also depressed GluR1-3 mRNA levels, suggesting that AMPAR down-regulation is a global response to extended periods of elevated neuronal activity. Analyses of time courses and thresholds indicated that BDNF expression is influenced by lower doses of, and shorter treatments with, the ampakine than is AMPAR expression. Accordingly, daily 3 h infusions of CX614 chronically elevated BDNF content with no effect on GluR1-3 concentrations. Restorative deconvolution microscopy provided the first evidence that chronic up-regulation of BDNF is accompanied by increased activation of the neurotrophin's TrkB-Fc receptor at spine synapses. These results show that changes in BDNF and AMPAR expression are dissociable and that up-regulation of the former leads to enhanced trophic signaling at excitatory synapses. These findings are encouraging with regard to the feasibility of using ampakines to tonically enhance BDNF-dependent functions in adult brain.Full Text Article
|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. |
Shilpa S Dhar, Huan Ling Liang, Margaret T T Wong-Riley
Journal of neurochemistry 108 1595-606 2009
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.
|The GluR2 subunit inhibits proliferation by inactivating Src-MAPK signalling and induces apoptosis by means of caspase 3/6-dependent activation in glioma cells. |
Beretta, Francesca, et al.
Eur. J. Neurosci., 30: 25-34 (2009) 2009
Glioblastoma multiforme (GBM) is the most invasive and undifferentiated type of brain tumour, and so surgical interventions are ineffective. We found that GluR2 is absent in fast-growing GBM-derived tumour stem cells and high-grade glioma specimens, but is expressed in slow-growing stem cells and low-grade glioma specimens. More remarkably, GluR2 overexpression in U-87MG cells inhibits proliferation by inactivating extracellular signal-regulated kinase (ERK)1/2-Src phosphorylation and induces apoptosis. Mechanistically, we observed that the scaffold protein GRIP is essential for the effect of GluR2 on ERK-Src inactivation. These findings indicate that the absence of the GluR2 subunit favours malignancy.
|Modulation of agonist binding to AMPA receptors by 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546): differential effects across brain regions and GluA1-4/transmembrane AMPA receptor regulatory prot |
Montgomery KE, Kessler M, Arai AC
The Journal of pharmacology and experimental therapeutics 331 965-74 2009
Ampakines are cognitive enhancers that potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents and synaptic responses by slowing receptor deactivation. Their efficacy varies greatly between classes of neurons and brain regions, but the factor responsible for this effect remains unclear. Ampakines also increase agonist affinity in binding tests in ways that are related to their physiological action. We therefore examined 1) whether ampakine effects on agonist binding vary across brain regions and 2) whether they differ across receptor subunits expressed alone and together with transmembrane AMPA receptor regulatory proteins (TARPs), which associate with AMPA receptors in the brain. We found that the maximal increase in agonist binding (E(max)) caused by the prototypical ampakine 1-(1,4-benzodioxan-6-ylcarbonyl)piperidine (CX546) differs significantly between brain regions, with effects in hippocampus and cerebellum being nearly three times larger than that in thalamus, brainstem, and striatum, and cortex being intermediate. These differences can be explained at least in part by regional variations in receptor subunit and TARP expression because combinations prevalent in hippocampus (GluA2 with TARPs gamma3 and gamma8) exhibited E(max) values nearly twice those of combinations abundant in thalamus (GluA4 with gamma2 or gamma4). TARPs seem to be critical because GluA2 and GluA4 alone had comparable E(max) and also because hippocampal and thalamic receptors had similar E(max) after solubilization with Triton X-100, which probably removes associated proteins. Taken together, our data suggest that variations in physiological drug efficacy, such as the 3-fold difference previously seen in recordings from hippocampus versus thalamus, may be explained by region-specific expression of GluA1-4 as well as TARPs.Full Text Article
|Increased AMPA receptor GluR1 subunit incorporation in rat hippocampal CA1 synapses during benzodiazepine withdrawal. |
Paromita Das, Scott M Lilly, Ricardo Zerda, William T Gunning, Francisco J Alvarez, Elizabeth I Tietz
The Journal of comparative neurology 511 832-46 2008
Prolonged benzodiazepine treatment leads to tolerance and increases the risk of dependence. Flurazepam (FZP) withdrawal is associated with increased anxiety correlated with increased alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPAR)-mediated synaptic function and AMPAR binding in CA1 pyramidal neurons. Enhanced AMPAR synaptic strength is also associated with a shift toward inward rectification of synaptic currents and increased expression of GluR1, but not GluR2, subunits, suggesting augmented membrane incorporation of GluR1-containing, GluR2-lacking AMPARs. To test this hypothesis, the postsynaptic incorporation of GluR1 and GluR2 subunits in CA1 neurons after FZP withdrawal was examined by postembedding immunogold quantitative electron microscopy. The percentage of GluR1 positively labeled stratum radiatum (SR) synapses was significantly increased in FZP-withdrawn rats (88.2% +/- 2.2%) compared with controls (74.4% +/- 1.9%). In addition, GluR1 immunogold density was significantly increased by 30% in SR synapses in CA1 neurons from FZP-withdrawn rats compared with control rats (FZP: 14.1 +/- 0.3 gold particles/mum; CON: 10.8 +/- 0.4 gold particles/mum). In contrast, GluR2 immunogold density was not significantly different between groups. Taken together with recent functional data from our laboratory, the current study suggests that the enhanced glutamatergic strength at CA1 neuron synapses during benzodiazepine withdrawal is mediated by increased incorporation of GluR1-containing AMPARs. Mechanisms underlying synaptic plasticity in this model of drug dependence are therefore fundamentally similar to those that operate during activity-dependent plasticity.Full Text Article
|Expression of AMPA receptor subunits at synapses in laminae I-III of the rodent spinal dorsal horn. |
Erika Polgár,Masahiko Watanabe,Bettina Hartmann,Seth Gn Grant,Andrew J Todd
Molecular pain 4 2008
Glutamate receptors of the AMPA type (AMPArs) mediate fast excitatory transmission in the dorsal horn and are thought to underlie perception of both acute and chronic pain. They are tetrameric structures made up from 4 subunits (GluR1-4), and subunit composition determines properties of the receptor. Antigen retrieval with pepsin can be used to reveal the receptors with immunocytochemistry, and in this study we have investigated the subunit composition at synapses within laminae I-III of the dorsal horn. In addition, we have compared staining of AMPArs with that for PSD-95, a major constituent of glutamatergic synapses. We also examined tissue from knock-out mice to confirm the validity of the immunostaining.Full Text Article
|Early-life epileptiform discharges exert both rapid and long-lasting effects on AMPAR subunit composition and distribution in developing neurons. |
Qian Jiang, Jingmin Wang, Ye Wu, Xiru Wu, Jiong Qin, Yuwu Jiang, Qian Jiang, Jingmin Wang, Ye Wu, Xiru Wu, Jiong Qin, Yuwu Jiang
Neuroscience letters 444 31-5 2008
The perinatal period of brain is characterized by dynamic changes in structure and high propensity for epilepsy. Animal models have shown that alterations of AMPA receptor (AMPAR) assembly or function may be related to seizure-induced cell damage, long-lasting impairments in brain development and seizure threshold. However, effects of earlier epileptiform discharges on AMPAR composition and sub-cellular distribution remain understudied. In this study, we analyzed age-dependent variation of relative GluR1 and GluR2 protein levels in primary cultured rat cortical neurons at 7DIV, 12DIV, 17DIV and 21DIV. By inducing a single event of epileptiform activity at 6DIV, we tested the effects of early-life seizure-like insults on AMPAR subunit distribution. We found a significant increase in synaptosomal membrane GluR1 expression in magnesium-free (MGF) medium-treated neurons at each time point detected (p0.05), while GluR2 expression increased at 7DIV, and declined at 17DIV and 21DIV respectively (p0.05). That is, a trend of high GluR1 with much lower GluR2 expression on the surface membrane of epileptiform discharges experienced neurons over time in culture was presented. These findings in an in vitro model of early-life seizure may inform rodent models of epilepsy, as well as the cellular mechanism involved in epilepsy-associated brain dysfunction.
|Long-term upregulation of protein kinase A and adenylate cyclase levels in human smokers. |
Bruce T Hope, Deepti Nagarkar, Sherry Leonard, Roy A Wise
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 1964-72 2007
Repeated injections of cocaine and morphine in laboratory rats cause a variety of molecular neuroadaptations in the cAMP signaling pathway in nucleus accumbens and ventral tegmental area. Here we report similar neuroadaptations in postmortem tissue from the brains of human smokers and former smokers. Activity levels of two major components of cAMP signaling, cAMP-dependent protein kinase A (PKA) and adenylate cyclase, were abnormally elevated in nucleus accumbens of smokers and in ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of the catalytic subunit of PKA were correspondingly higher in the ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of other candidate neuroadaptations, including glutamate receptor subunits, tyrosine hydroxylase, and other protein kinases, were within normal range. These findings extend our understanding of addiction-related neuroadaptations of cAMP signaling to tobacco smoking in human subjects and suggest that smoking-induced brain neuroadaptations can persist for significant periods in former smokers.Full Text Article
|OFF midget bipolar cells in the retina of the marmoset, Callithrix jacchus, express AMPA receptors. |
Christian Puller,Silke Haverkamp,Ulrike Grünert
The Journal of comparative neurology 502 2007
Recent studies suggested that different types of OFF bipolar cells express specific types of ionotropic (AMPA or kainate) glutamate receptors (GluRs) at their contacts with cone pedicles. However, the question of which GluR type is expressed by which type of OFF bipolar cell in primate retina is still open. In this study, the expression of AMPA and kainate receptor subunits at the dendritic tips of flat (OFF) midget bipolar (FMB) cells was analyzed in the retina of the common marmoset, Callithrix jacchus. We used preembedding electron microscopy and double immunofluorescence with subunit-specific antibodies. The FMB cells were labeled with antibodies against the carbohydrate epitope CD15. Cone pedicles were identified with peanut agglutinin. Immunoreactivity for the GluR1 subunit and for CD15 is preferentially located at triad-associated flat contacts. Furthermore, the large majority of GluR1 immunoreactive puncta is localized at the dendritic tips of FMB cells. These results suggest that FMB cells express the AMPA receptor subunit GluR1. In contrast, the kainate receptor subunit GluR5 is not colocalized with the dendritic tips of FMB cells or with the GluR1 subunit. Immunoreactive puncta for the GluR1 subunit are found at all M/L-cone pedicles but are only rarely associated with S-cone pedicles. This is consistent with our recent findings in marmoset retina that FMB cells do not contact S-cone pedicles. The presence of GluR5 clusters at S-cone pedicles indicates that in primate retinas OFF bipolar cells expressing kainate receptor subunits receive some S-cone input.
|Distribution of AMPA glutamate receptor GluR1 subunit-immunoreactive neurons and their co-localization with calcium-binding proteins and GABA in the mouse visual cortex. |
Kim, Tae-Jin, et al.
Mol. Cells, 21: 34-41 (2006) 2006
The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.
|Synaptic pattern of AMPA receptor subtypes upon direction-selective retinal ganglion cells. |
Jeong, Seong-Ah, et al.
Neurosci. Res., 56: 427-34 (2006) 2006
In the search for anisotropies that might contribute to a directional preference of direction-selective (DS) retinal ganglion cells (RGCs), we studied the distributions of AMPA receptor subtypes GluR1, GluR2/3, and GluR4 upon the dendritic arbors of DS RGCs of the rabbit with antibody immunocytochemistry. DS RGCs were injected with Lucifer yellow and the cells were identified by their characteristic morphology. The double-labeled images of dendrites and receptors were visualized by confocal microscopy and were reconstructed from high-resolution confocal images. We found no evidence of asymmetry in any of the AMPA receptor subunits examined upon the dendritic arbors of both On and Off layers of DS RGCs. The present results indicate that direction selectivity appears to lie in presynaptic pattern.
|Ionotropic Glutamate Receptor GluR1 in the Visual Cortex of Hamster: Distribution and Co-Localization with Calcium-Binding Proteins and GABA. |
Ye, Eun-Ah, et al.
Acta histochemica et cytochemica, 39: 47-54 (2006) 2006
|An AMPA glutamatergic receptor activation-nitric oxide synthesis step signals transsynaptic apoptosis in limbic cortex. |
Yueping Zhou, Lijun Zhou, Haiming Chen, Vassilis E Koliatsos
Neuropharmacology 51 67-76 2006
We have previously shown that pyramidal neurons engaged in cortico-cortical connectivity in limbic cortex are vulnerable to denervation lesions, i.e. relay pyramidal neurons in layer II of piriform cortex undergo transsynaptic apoptosis after lesions interrupting their inputs from the olfactory bulb (bulbotomies). At least one trigger of this transsynaptic degenerative phenomenon is the activation of inhibitory interneurons in layer I, which are induced to upregulate neuronal nitric oxide synthase (nNOS) and release NO. Thus, we have demonstrated that cortical interneurons play an essential role in transducing injury to apoptotic signaling that selectively targets pyramidal neurons. In the present study, we confirm the role of nNOS with pharmacological inhibition of a significant approximately 30% of transsynaptic apoptosis with the selective nNOS inhibitor BRNI at optimal doses. Outcomes were studied both at the histological and molecular level using DNA blots. We also show that the first-generation competitive non-NMDA (AMPA) antagonist NBQX ameliorates transsynaptic apoptosis by the same margin of difference as BRNI and it also reduces nNOS activation as indicated by a significant decrease in NADPH diaphorase histochemical activity in layer I of piriform cortex. Our findings confirm the role of nNOS activation/NO release in transsynaptic apoptosis and show that glutamatergic agonism at AMPA sites also plays a significant role. In addition, our data suggest that AMPA agonism may occur upstream to nNOS upregulation in inhibitory interneurons of layer I. In concert, our findings indicate that transsynaptic neuronal degeneration in limbic cortex involves complex AMPA-glutamatergic and nitrinergic signaling events. An AMPA-mediated upregulation of nNOS and release of NO by inhibitory interneurons may play a prominent role in this type of injury.
|Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography. |
Philippe Rostaing, Eleonore Real, Léa Siksou, Jean-Pierre Lechaire, Thomas Boudier, Tobias M Boeckers, Frank Gertler, Eckart D Gundelfinger, Antoine Triller, Serge Marty
The European journal of neuroscience 24 3463-74 2006
Electron microscopy allows the analysis of synaptic ultrastructure and its modifications during learning or in pathological conditions. However, conventional electron microscopy uses aldehyde fixatives that alter the morphology of the synapse by changing osmolarity and collapsing its molecular components. We have used high-pressure freezing (HPF) to capture within a few milliseconds structural features without aldehyde fixative, and thus to provide a snapshot of living synapses. CA1 hippocampal area slices from P21 rats were frozen at -173 degrees C under high pressure to reduce crystal formation, and synapses on dendritic spines were analysed after cryosubstitution and embedding. Synaptic terminals were larger than after aldehyde fixation, and synaptic vesicles in these terminals were less densely packed. Small filaments linked the vesicles in subgroups. The postsynaptic densities (PSDs) exhibited filamentous projections extending into the spine cytoplasm. Tomographic analysis showed that these projections were connected with the spine cytoskeletal meshwork. Using immunocytochemistry, we found as expected GluR1 at the synaptic cleft and CaMKII in the PSD. Actin immunoreactivity (IR) labelled the cytoskeletal meshwork beneath the filamentous projections, but was very scarce within the PSD itself. ProSAP2/Shank3, cortactin and Ena/VASP-IRs were concentrated on the cytoplasmic face of the PSD, at the level of the PSD projections. Synaptic ultrastructure after HPF was different from that observed after aldehyde fixative. The boutons were larger, and filamentous components were preserved. Particularly, filamentous projections were observed linking the PSD to the actin cytoskeleton. Thus, synaptic ultrastructure can be analysed under more realistic conditions following HPF.
|Comprehensive identification of phosphorylation sites in postsynaptic density preparations. |
Jonathan C Trinidad, Christian G Specht, Agnes Thalhammer, Ralf Schoepfer, Alma L Burlingame
Molecular cellular proteomics : MCP 5 914-22 2006
In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.
|The role of AMPA receptor gating in the development of high-fidelity neurotransmission at the calyx of Held synapse. |
Joshi, Indu, et al.
J. Neurosci., 24: 183-96 (2004) 2004
During early postnatal development of auditory synapses, the decay time course of AMPA receptor (AMPAR) EPSCs accelerates markedly, but the mechanisms underlying this process remain uncertain. Using the developing calyx of Held synapse in the mouse auditory brainstem, we have examined presynaptic and postsynaptic elements that may regulate decay kinetics of AMPAR EPSCs. We found that the decay time kinetics was voltage dependent in both immature and mature synapses, being slower at positive potentials than negative potentials. By recording evoked miniature events in extracellular Ca2+ or Sr2+, we revealed a significant decrease in decay time constants of EPSCs as maturation progresses. On the basis of internal and external polyamine block of AMPAR EPSCs and immunohistochemistry assays with subunit-specific antibodies, we demonstrated that the glutamate receptor (GluR) 2 subunit is virtually absent at all developmental ages. Antibody staining patterns suggest a gradual shift in subunit composition from GluR1- to GluR3/4-dominant phenotypes. Kinetic analyses of deactivation, desensitization, and recovery from desensitization in outside-out patches in response to ultrafast application of glutamate lend supportive evidence that such a shift in the gating phenotype likely accounts for the accelerated time course throughout development. Finally, by pharmacologically manipulating AMPAR gating and using simulated EPSCs to evoke action potentials, we demonstrated that rapid decay kinetics of AMPAR EPSCs is essential for this synapse to accommodate high-frequency firing without compromising spike amplitude. Hence, developmental alterations in the subunit composition likely dictate changes in the time course of AMPAR EPSCs and play an indispensable role in the refinement of high-fidelity neurotransmission at the calyx of Held synapse.
|Postsynaptic density assembly is fundamentally different from presynaptic active zone assembly. |
Bresler, Tal, et al.
J. Neurosci., 24: 1507-20 (2004) 2004
The cellular mechanisms involved in the formation of the glutamatergic postsynaptic density (PSD) are mainly unknown. Previous studies have indicated that PSD assembly may occur in situ by a gradual recruitment of postsynaptic molecules, whereas others have suggested that the PSD may be assembled from modular transport packets assembled elsewhere. Here we used cultured hippocampal neurons and live cell imaging to examine the process by which PSD molecules from different layers of the PSD are recruited to nascent postsynaptic sites. GFP-tagged NR1, the essential subunit of the NMDA receptor, and ProSAP1/Shank2 and ProSAP2/Shank3, scaffolding molecules thought to reside at deeper layers of the PSD, were recruited to new synaptic sites in gradual manner, with no obvious involvement of discernible discrete transport particles. The recruitment kinetics of these three PSD molecules were remarkably similar, which may indicate that PSD assembly rate is governed by a common upstream rate-limiting process. In contrast, the presynaptic active zone (AZ) molecule Bassoon was observed to be recruited to new presynaptic sites by means of a small number of mobile packets, in full agreement with previous studies. These findings indicate that the assembly processes of PSDs and AZs may be fundamentally different.
|Increased expression of AMPA receptor subunits in the nucleus of the solitary tract in the spontaneously hypertensive rat. |
Saha, Sikha, et al.
Brain Res. Mol. Brain Res., 121: 37-49 (2004) 2004
The expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits GluR1-4 in the nucleus of the solitary tract (NTS) of adult Wistar rats was examined by polymerase chain reaction (PCR), and the neuronal localisation of these receptor subunits in the NTS were confirmed by immunohistochemistry using subunit-specific antibodies. Semi-quantitative PCR was used to investigate differences in AMPA receptor subunit expression between spontaneously hypertensive rats (SH) and age-matched normotensive Wistar Kyoto rats (WKY). All four receptor subunits were expressed in both strains, but compared to WKY, total AMPA receptor and the GluR3 mRNA expressions were significantly higher in SH. No differences were detected in cDNA form the cerebral cortex or cerebellum. Immunolabelling for GluRs 1, 2 and 2/3 in the neuropil relative to neuronal somata in the cardioregulatory areas of the NTS appeared to be increased in SH, with an overall increase in the density of GluR2/3 labelling in the medial and commissural NTS of SH. These results indicate a possible role for changes in AMPA receptor subunit expression in NTS neurones, involving an increase in GluR3 associated with development of hypertension in SH.
|Neuronal pentraxin 1: a novel mediator of hypoxic-ischemic injury in neonatal brain. |
Hossain, Mir Ahamed, et al.
J. Neurosci., 24: 4187-96 (2004) 2004
Neonatal hypoxic-ischemic brain injury is a major cause of neurological disability and mortality. Its therapy will likely require a greater understanding of the discrete neurotoxic molecular mechanism(s) triggered by hypoxia-ischemia (HI). Here, we investigated the role of neuronal pentraxin 1 (NP1), a member of a newly recognized subfamily of "long pentraxins," in the HI injury cascade. Neonatal brains developed marked infarcts in the ipsilateral cerebral hemisphere at 24 hr and showed significant loss of ipsilateral striatal, cortical, and hippocampal volumes at 7 d after HI compared with the contralateral hemisphere and sham controls. Immunofluorescence analyses revealed elevated neuronal expression of NP1 in the ipsilateral cerebral cortex from 6 hr to 7 d and in the hippocampal CA1 and CA3 regions from 24 hr to 7 d after HI. These same brain areas developed infarcts and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells within 24-48 hr of HI. In primary cortical neurons, NP1 protein was induced >2.5-fold (p < 0.001) after their exposure to hypoxia that caused approximately 30-40% neuronal death. Transfecting cortical neurons with antisense oligodeoxyribonucleotides directed against NP1 mRNA (NP1AS) significantly inhibited (p < 0.01) hypoxia-induced NP1 protein induction and neuronal death (p < 0.001), demonstrating a specific requirement of NP1 in hypoxic neuronal injury. NP1 protein colocalized and coimmunoprecipitated with the fast excitatory AMPA glutamate receptor subunit (GluR1) in primary cortical neurons, and hypoxia induced a time-dependent increase in NP1-GluR1 interactions. NPIAS also protected against AMPA-induced neuronal death (p < 0.05), implicating a role for NP1 in the excitotoxic cascade. Our results show that NP1 induction mediates hypoxic-ischemic injury probably by interacting with and modulating GluR1 and potentially other excitatory glutamate receptors.
|Independent functions of hsp90 in neurotransmitter release and in the continuous synaptic cycling of AMPA receptors. |
Gerges, Nashaat Z, et al.
J. Neurosci., 24: 4758-66 (2004) 2004
The delivery of neurotransmitter receptors into synapses is essential for synaptic function and plasticity. In particular, AMPA-type glutamate receptors (AMPA receptors) reach excitatory synapses according to two distinct routes: a regulated pathway, which operates transiently during synaptic plasticity, and a constitutive pathway, which maintains synaptic function under conditions of basal transmission. However, the specific mechanisms that distinguish these two trafficking pathways are essentially unknown. Here, we evaluate the role of the molecular chaperone hsp90 (heat shock protein 90) in excitatory synaptic transmission in the hippocampus. On one hand, we found that hsp90 is necessary for the efficient neurotransmitter release at the presynaptic terminal. In addition, we identified hsp90 as a critical component of the cellular machinery that delivers AMPA receptors into the postsynaptic membrane. Using the hsp90-specific inhibitors radicicol and geldanamycin, we show that hsp90 is required for the constitutive trafficking of AMPA receptors into synapses during their continuous cycling between synaptic and nonsynaptic sites. In contrast, hsp90 function is not required for either the surface delivery of AMPA receptors into the nonsynaptic plasma membrane or for the acute, regulated delivery of AMPA receptors into synapses during plasticity induction (long-term potentiation). The synaptic cycling of AMPA receptors was also blocked by an hsp90-binding tetratricopeptide repeat (TPR) domain, suggesting that the role of hsp90 in AMPA receptor trafficking is mediated by a TPR domain-containing protein. These results demonstrate new roles for hsp90 in synaptic function by controlling neurotransmitter release and, independently, by mediating the continuous cycling of synaptic AMPA receptors.
|Spinal axonal injury induces brief downregulation of ionotropic glutamate receptors and no stripping of synapses in cord-projection central neurons. |
Yueh-Jan Wang, Guo-Fang Tseng
Journal of neurotrauma 21 1624-39 2004
Spinal cord injury often damages the axons of cord-projecting central neurons. To determine whether their excitatory inputs are altered following axonal injury, we used rat rubrospinal neurons as a model and examined their excitatory input following upper cervical axotomy. Anterograde tracing showed that the primary afferents from the cerebellum terminated in a pattern similar to that of control animals. Ultrastructurally, neurons in the injured nucleus were contacted by excitatory synapses of normal appearance, with no sign of glial stripping. Since cerebellar fibers are glutamatergic, we examined the expression of ionotropic receptor subunits GluR1-4 and NR1 for AMPA and NMDA receptors, respectively, in control and injured neurons using immunolabeling methods. In control neurons, GluR2 appeared to be low as compared to GluR1, GluR3, and GluR4, while NR1 labeling was intense. Following unilateral tractotomy, the levels of expression of each subunit in axotomized neurons appeared to be normal, with the exception that they were lower than those of control neurons of the nonlesioned side at 2-6 days postinjury. These findings suggest that axotomized neurons are only temporarily protected from excitotoxicity. This is in sharp contrast to the responses of central neurons that innervate peripheral targets, in which both synaptic stripping and reduction of their ionotropic glutamate receptor subunits persist following axotomy. The absence of an injury-induced trimming of afferents and stripping of synapses and the lack of a persistent downregulation of postsynaptic receptors might enable injured cord-projection neurons to continue to control their supraspinal targets during most of their postinjury survival. Although this may support neurons by providing trophic influences, it nevertheless may subject them to excitotoxicity and ultimately lead to their degenerative fate.
|Differential expression of NMDA and AMPA receptor subunits in DARPP-32-containing neurons of the cerebral cortex, hippocampus and neostriatum of rats. |
W-W Wang, R Cao, Z-R Rao, L-W Chen
Brain research 998 174-83 2004
Dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32) is a key element of dopamine/D1/DARPP-32/protein phosphatase-1 (PP-1) signaling cascades of mammalian brain. We are interested in the expression patterns of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in DARPP-32-containing neurons, which may constitute morphological basis for interaction between dopamine and ionotropic glutamate receptors in dopaminoceptive cells. Double immunofluorescence was performed to visualize neurons showing coexpression of DARPP-32 with NMDA or AMPA receptor subunits (i.e., NR1, NR2a/b, glutamate receptor subunit 1 [GluR1], GluR2/3, and GluR4) in the forebrains of rats. Distribution of DARPP-32-positive neurons completely or partially overlapped with that of NMDA receptor- or AMPA receptor-immunoreactive ones in the frontal and parietal cortex, hippocampus and neostriatum, and neurons double-labeled with DARPP-32/NR1, DARPP-32/NR2a/b, DARPP-32/GluR1, DARPP-32/GluR2/3, or DARPP-32/GluR4 immunoreactivity were numerously observed. Semiquantification analysis indicated that most of DARPP-32-containing neurons (86-98%) expressed NR1, NR2a/b and GluR2/3, while less of them (14-90%) expressed GluR1 and GluR4. Although high rates (90-98%) of DARPP-32-positive cells expressed NMDA receptors in all regions above, variant percentages of them expressing AMPA receptor subunits were observed among the cortex (54-90%), hippocampus (59-97%) and neostriatum (14-97%). The study presents differential expression patterns of NMDA and AMPA receptors in DARPP-32-postive neurons in these forebrain regions. Taken together with previous reports, the present data suggest that interaction between dopamine and glutamate receptors may occur in the dopaminoceptive neurons with distinct receptor compositions and may be involved in modulating neuronal properties and excitotoxicity in mammalian forebrain.
|Alterations of markers related to synaptic function in aging rat brain, in normal conditions or under conditions of long-term dietary manipulation. |
Barbara Monti, Marco Virgili, Antonio Contestabile
Neurochemistry international 44 579-84 2004
Neurochemical alterations of markers related to synaptic function are potential candidates for age-related impairment of brain function and cognition. The process of aging, including brain aging, can be counteracted to some degree by maintaining animals in long-term conditions of caloric restriction, or supplementing their diet with antioxidant substances. We report here that the age-related decline of the cholinergic and GABAergic systems, that takes place in some CNS regions of aged rats, is not affected by maintaining them under conditions of dietary restriction and, therefore, of reduced calorie intake, from the 12th to the 30th month of age. We also notice the same lack of effect by adding, during the same period, the aging rat diet with the potential antioxidant substance, N-acetylcysteine (NAC). The same dietary manipulations are also unable to counteract the derangement of the first step of the main biosynthetic pathway for polyamines, putative neuromodulators in the CNS, that occurs in the aged spinal cord. Some age-related alterations in the expression of different subunits of the NMDA-type glutamate receptors in some CNS regions of aged rats were instead, at least in some cases, counteracted by long-term dietary manipulation.
|Synaptic glutamate receptor clustering in mice lacking the SH3 and GK domains of SAP97. |
Klöcker, Nikolaj, et al.
Eur. J. Neurosci., 16: 1517-22 (2002) 2002
Postsynaptic targeting of the Drosophila tumour suppressor discs-large (Dlg) critically depends on its SH3 and GK domains. Here, we asked whether these domains are also involved in subcellular targeting of the mammalian Dlg homolog SAP97 and its interacting partners in CNS cortical neurons by analysing a recently described mouse mutant lacking the SH3 and GK domains of SAP97. Both wildtype and truncated SAP97 were predominantly expressed in perinuclear regions, in a pattern suggesting association with the endoplasmic reticulum. Weaker immunoreactivity was found in neurites colocalizing with both dendritic and axonal markers. As SAP97 has been implicated in the early intracellular processing of the glutamate receptor GluR1, we studied biochemical maturation and subcellular localization of GluR1 in the mutants. Both the glycosylation pattern and synaptic clustering of GluR1 were indistinguishable from wildtype mice. Synaptic clustering of the guanylate kinase domain interacting protein GKAP was also intact. Our data demonstrate that truncation of the SH3 and GK domains of SAP97 in mice does neither change its subcellular distribution nor does it disrupt synaptic structure or protein clustering, as opposed to severe missorting of the respective mutant Dlg protein in Drosophila.