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|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.
|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.
|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.
|Differences in ionotropic glutamate receptor subunit expression are not responsible for strain-dependent susceptibility to excitotoxin-induced injury. |
Schauwecker, Paula Elyse
Brain Res. Mol. Brain Res., 112: 70-81 (2003) 2003
Systemic administration of kainic acid in C57BL/6 and FVB/N mice induces a comparable level of seizure induction yet results in differential susceptibility to seizure-induced cell death. While kainate administration causes severe hippocampal damage in mice of the FVB/N strain, C57BL/6 mice display no demonstrable cell loss or damage. At present, while the cellular mechanisms underlying strain-dependent differences in susceptibility remain unclear, some of this variation is assumed to have a genetic basis. As glutamate receptors are thought to participate in seizure induction and the subsequent neuronal degeneration that ensues, previous studies have proposed that variation in the precise subunit composition of glutamate receptors may result in differential susceptibility to excitotoxic cell death. Thus, we chose to examine the relationship between the cellular distribution and expression of glutamate receptor subunit proteins and cell loss within the hippocampus in mouse strains resistant and susceptible to kainate-induced excitotoxicity. Using semi-quantitative Western blot techniques and immunohistochemistry with the use of antibodies that recognize subunits of the KA (GluR5,6,7), AMPA (GluR1, GluR2, and GluR4), and NMDA (NMDAR1 and NMDAR2A/2B) receptors, we found no significant strain-dependent differences in the expression or distribution of these glutamate receptor subunits in the intact hippocampus. Following kainate administration, expression changes in ionotropic glutamate receptor subunits paralleled the development of susceptibility to cell death in the FVB/N strain only. Strain differences in hippocampal vulnerability to kainate-induced status epilepticus are not due to glutamate receptor protein expression.
|Ca(2+)-evoked synaptic transmission and neurotransmitter receptor levels are impaired in the forebrain of trkb (-/-) mice. |
Carmona, Maria A, et al.
Mol. Cell. Neurosci., 22: 210-26 (2003) 2003
|Ionotropic glutamate receptor subunit immunoreactivity of vagal preganglionic neurones projecting to the rat heart. |
Corbett, Eric K A, et al.
Auton Neurosci, 105: 105-17 (2003) 2003
The ionotropic glutamate receptor subunits expressed by vagal preganglionic neurones in the rat medulla oblongata were examined by using fluorescence immunolabelling combined with retrograde neuronal tracing. The general population of these neurones in the medulla was identified by intraperitoneal injections of Fluorogold and also with choline acetyltransferase antibodies. Cardiac projecting neurones were specifically identified by applying the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (DiI) to the heart or by injecting cholera toxin B-subunit into the pericardium. Both tracers labelled populations of neurones lying in the dorsal vagal nucleus, intermediate reticular formation and nucleus ambiguus, and when both tracers were applied simultaneously, approximately 50% of cells were dual-labelled. Control experiments established that the labelling was specific for neurones projecting to the heart. Most vagal preganglionic neurones, including those projecting to the heart, irrespective of their location in the medulla, had a similar profile of glutamate receptor immunoreactivity. Labelling of somata for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) subunit GluR1 was weak or absent, while labelling with antibodies directed to GluR2, a common sequence of GluR2 and GluR3, and GluR4 was moderate or intense. All neurones studied appeared to express the N-methyl-D-aspartate (NMDA) receptor subunit NR1, and while antibodies recognising the NR2A and NR2B splice variants gave strong labelling, immunoreactivity with a NR2B specific antibody was weaker. Weak to moderate labelling was seen in some neurones using antibodies to the kainate receptor subunits KA2 and GluR5-7. These results are consistent with neurophysiological data indicating the presence of AMPA, NMDA and kainate responses in cardiac vagal preganglionic neurones, and suggest that these neurones are similar to other vagal parasympathetic preganglionic neurones in expressing mainly AMPA receptor subunits GluR2-4.
|Characterization of the intracellular transport of GluR1 and GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits in hippocampal neurons. |
Pavel V Perestenko, Jeremy M Henley
The Journal of biological chemistry 278 43525-32 2003
Little is known about the dynamics of the dendritic transport of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) to synapses. Here, using virally expressed green fluorescent protein (GFP)-GluR1 and GFP-GluR2 and confocal photobleach techniques we show near real-time movement of these subunits in living cultured hippocampal neurons. GFP-GluR1 fluorescence was widely distributed throughout the extranuclear compartment with no evidence for discrete intracellular stores. GFP-GluR1 transport was predominantly proximal to distal at rates of 0.2-0.4 mum.s-1. GFP-GluR2 fluorescence was more punctate and localized at or close to the plasma membrane. Overall, GFP-GluR2 movement was less dynamic with distinct mobile and immobile pools. Neither activation nor inhibition of surface-expressed N-methyl-d-aspartate receptors or AMPARs had any significant effect on the rates of GFP-GluR1 or GFP-GluR2 dendritic transport. These results demonstrate that GluR1 is constitutively and rapidly transported throughout the neuron. GluR2, on the other hand, is less mobile, with a majority retained in relatively immobile membrane-associated clusters, with approximately 40% showing synaptic co-localization. Furthermore, the transport of both subunits is activity-independent, suggesting that the regulated delivery of AMPARs to the vicinity of synapses is not a mechanism that is involved in processes such as synaptic plasticity.
|Absence of Whisker-related pattern formation in mice with NMDA receptors lacking coincidence detection properties and calcium signaling. |
York Rudhard, Matthias Kneussel, Mohammed A Nassar, Georg F Rast, Alexander J Annala, Philip E Chen, Cezar M Tigaret, Isabel Dean, Juergen Roes, Alasdair J Gibb, Stephen P Hunt, Ralf Schoepfer
The Journal of neuroscience : the official journal of the Society for Neuroscience 23 2323-32 2003
Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+ impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NMDAR subunits and other important neurotransmitter receptors. Our results show that coincidence detection by, and/or Ca2+ permeability of, NMDARs is necessary for the development of somatotopic maps in the brainstem and suggest that highly specific signaling underlies synaptic refinement.
|Light microscopic study of GluR1 and calbindin expression in interneurons of neocortical microgyral malformations. |
V N Kharazia, K M Jacobs, D A Prince
Neuroscience 120 207-18 2003
Rat neocortex that has been injured on the first or second postnatal day (P0-1) develops an epileptogenic, aberrantly layered malformation called a microgyrus. To investigate the effects of this developmental plasticity on inhibitory interneurons, we studied a sub-population of GABAergic cells that co-express the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit and the calcium-binding protein, calbindin (CB). Both malformed and control cortex of adult (P40-60) animals contained numerous interneurons double-stained for CB and GluR1. Immunoreactivity (IR) for CB was up-regulated in perikarya of interneurons within supragranular layers of control cortex between P12 and P40. However, in malformed adult (P40) cortex, CB-IR levels were significantly lower than in adult controls, and fell midway between levels in immature and adult control animals. Between P12 and P40, GluR1-IR was down-regulated in perikarya of interneurons in control cortex. Somatic GluR1-IR levels in malformed adult (P40) cortex were not different from adult controls. These neurons formed a dense plexus of highly GluR1-positive spiny dendrites within layer II. The dendritic plexus in the malformation was more intensely GluR1-immunoreactive than that in layer II of control cortex. This was due to apparent changes in thickness and length of dendrites, rather than to significant changes in the number of interneuronal perikarya in the microgyral cortex. Results indicate that the population of GluR1/CB-containing interneurons is spared in malformed microgyral cortex, but that these cells sustain lasting decreases in their somatic expression of calbindin and alterations of dendritic structure. Potential functional implications of these findings are discussed.
|Ligand-independent CXCR2 dimerization. |
Flavia Trettel, Sabrina Di Bartolomeo, Clotilde Lauro, Myriam Catalano, Maria Teresa Ciotti, Cristina Limatola
The Journal of biological chemistry 278 40980-8 2003
Homo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product beta) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.
|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.
|Functional expression of AMPA receptors on central terminals of rat dorsal root ganglion neurons and presynaptic inhibition of glutamate release. |
Lee, C Justin, et al.
Neuron, 35: 135-46 (2002) 2002
No direct evidence has been found for expression of functional AMPA receptors by dorsal root ganglion neurons despite immunocytochemical evidence suggesting they are present. Here we report evidence for expression of functional AMPA receptors by a subpopulation of dorsal root ganglion neurons. The AMPA receptors are most prominently located near central terminals of primary afferent fibers. AMPA and kainate receptors were detected by recording receptor-mediated depolarization of the central terminals under selective pharmacological conditions. We demonstrate that activation of presynaptic AMPA receptors by exogenous agonists causes inhibition of glutamate release from the terminals, possibly via primary afferent depolarization (PAD). These results challenge the traditional view that GABA and GABA(A) receptors exclusively mediate PAD, and indicate that PAD is also mediated by glutamate acting on presynaptically localized AMPA and kainate receptors.
|Enrichment of N-methyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities. |
Al-Hallaq, R A, et al.
J. Neurochem., 77: 110-9 (2001) 2001
Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.
|Nicotine receptor inactivation decreases sensitivity to cocaine. |
Zachariou, V, et al.
Neuropsychopharmacology, 24: 576-89 (2001) 2001
The reinforcing properties of nicotine and psychomotor stimulants are thought to be mediated through the mesolimbic dopamine (DA) system. This study investigates the role of high affinity nicotinic acetylcholine receptors (nAChRs) in cocaine place preference and examines some neurochemical changes in the mesolimbic DA system that might account for the interaction between nicotine and cocaine. 5 mg/kg is the lowest dose of cocaine able to condition a place preference in C57Bl/6 mice. Co-treatment with the nicotinic antagonist mecamylamine (1.0 mg/kg) disrupted place preference to 5 mg/kg cocaine. In addition, mice lacking the high affinity nAChR containing the beta2 subunit showed decreased place preference to 5 mg/kg cocaine, although higher doses of cocaine could condition a place preference in these knock out animals. In contrast, co-administration of a low dose of nicotine (0.2 mg/kg) potentiated place preference to a subthreshold dose of cocaine (3 mg/kg). DA turnover was monitored in several brain regions using tissue levels of DA and its primary metabolite DOPAC as an indication of DA release. Wild type mice showed decreased DA turnover following treatment with 5 mg/kg cocaine; whereas, this response was not seen in mice lacking the beta2 subunit of the nAChR. Induction of chronic fos-related antigens by cocaine was also reduced in mutant mice as compared to their wild type siblings, implying that downstream actions of cocaine were also affected by inactivation of the high affinity nAChR. These data indicate that activation of the high affinity nAChR may contribute to cocaine reinforcement.
|Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. |
Cherny, R A, et al.
Neuron, 30: 665-76 (2001) 2001
Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimer's disease (AD). Cu and Zn are enriched in Abeta deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. Here we report a 49% decrease in brain Abeta deposition (-375 microg/g wet weight, p = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks with clioquinol, an antibiotic and bioavailable Cu/Zn chelator. This was accompanied by a modest increase in soluble Abeta (1.45% of total cerebral Abeta); APP, synaptophysin, and GFAP levels were unaffected. General health and body weight parameters were significantly more stable in the treated animals. These results support targeting the interactions of Cu and Zn with Abeta as a novel therapy for the prevention and treatment of AD.
|Colocalization of GluR1 and neuronal nitric oxide synthase in rat nucleus tractus solitarii neurons. |
Lin, L H and Talman, W T
Neuroscience, 106: 801-9 (2001) 2001
Previously we demonstrated that glutamate and neuronal nitric oxide synthase (nNOS) containing neuronal elements are frequently apposed in subnuclei of the rat nucleus tractus solitarii. It is known that glutamate receptors (GluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype participate in cardiovascular regulation by the nucleus tractus solitarii and that responses to AMPA receptor activation may be linked to NO. Therefore, in the present study, we further tested the hypothesis that the calcium-permeable subunit GluR1 of AMPA type GluRs and nNOS are colocalized in neurons of the nucleus tractus solitarii. Distribution of GluR1 and nNOS in rat nucleus tractus solitarii was investigated by double fluorescent immunohistochemistry combined with confocal laser scanning microscopy.Numerous GluR1 immunoreactive cells and fibers were present in subnuclei of the nucleus tractus solitarii. The staining intensity of GluR1 immunoreactive cells varied among subnuclei. Cells in the interstitial subnucleus contained the highest GluR1 staining intensity. A moderate intensity of staining was present in the intermediate, dorsolateral, ventral, and commissural subnuclei. A slightly lower level of GluR1 immunoreactivity was present in cells of the medial subnucleus. Cells in the central subnucleus contained a low level of GluR1 immunoreactivity. The staining intensity of GluR1 immunoreactive fibers also varied among subnuclei. Distribution of nNOS immunoreactivity in the nucleus tractus solitarii and other brain stem areas was the same as in our earlier reports. Superimposition of confocal images of nNOS immunoreactivity and GluR1 immunoreactivity allowed us to identify double-labeled structures. Nearly all neurons that were immunoreactive for nNOS contained GluR1 immunoreactivity, but only a proportion of GluR1 immunoreactive cells contained nNOS immunoreactivity. Double-labeled neurons were present in all subnuclei of the nucleus tractus solitarii. The percentages of GluR1 immunoreactive cells that also contained nNOS immunoreactivity differed among subnuclei of the nucleus tractus solitarii. Fibers that labeled for nNOS alone, GluR1 alone or both were present among labeled cells in these subnuclei.These data support the hypothesis that GluR1 and nNOS are colocalized in neurons of nucleus tractus solitarii. The demonstration of this anatomical relationship provides further anatomical support for the hypothesis that activation of AMPA receptors on neurons that synthesize NO in the nucleus tractus solitarii contributes to autonomic regulation.
|Postmortem brain abnormalities of the glutamate neurotransmitter system in autism. |
A E Purcell, O H Jeon, A W Zimmerman, M E Blue, J Pevsner
Neurology 57 1618-28 2001
BACKGROUND: Studies examining the brains of individuals with autism have identified anatomic and pathologic changes in regions such as the cerebellum and hippocampus. Little, if anything, is known, however, about the molecules that are involved in the pathogenesis of this disorder. OBJECTIVE: To identify genes with abnormal expression levels in the cerebella of subjects with autism. METHOD: Brain samples from a total of 10 individuals with autism and 23 matched controls were collected, mainly from the cerebellum. Two cDNA microarray technologies were used to identify genes that were significantly up- or downregulated in autism. The abnormal mRNA or protein levels of several genes identified by microarray analysis were investigated using PCR with reverse transcription and Western blotting. alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)- and NMDA-type glutamate receptor densities were examined with receptor autoradiography in the cerebellum, caudate-putamen, and prefrontal cortex. RESULTS: The mRNA levels of several genes were significantly increased in autism, including excitatory amino acid transporter 1 and glutamate receptor AMPA 1, two members of the glutamate system. Abnormalities in the protein or mRNA levels of several additional molecules in the glutamate system were identified on further analysis, including glutamate receptor binding proteins. AMPA-type glutamate receptor density was decreased in the cerebellum of individuals with autism (p 0.05). CONCLUSIONS: Subjects with autism may have specific abnormalities in the AMPA-type glutamate receptors and glutamate transporters in the cerebellum. These abnormalities may be directly involved in the pathogenesis of the disorder.
|The human temporal cortex: characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles. |
M C González-Albo, G N Elston, J DeFelipe
Cerebral cortex (New York, N.Y. : 1991) 11 1170-81 2001
Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CR), in the inferotemporal gyrus (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, CB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal cells, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.
|Ionotropic glutamate receptors during the development of the chick retina. |
A Silveira dos Santos Bredariol, D E Hamassaki-Britto, A Silveira dos Santos Bredariol, D E Hamassaki-Britto
The Journal of comparative neurology 441 58-70 2001
Glutamate is the main neurotransmitter of photoreceptors, bipolar cells, and ganglion cells of the vertebrate retina. Three main classes of ionotropic glutamate receptors comprising different subunits can be distinguished: AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionate), KA (kainate), and NMDA (N-methyl-D-aspartate). This study was undertaken to characterize the AMPA (GluR1, GluR2/3, and GluR4), KA (GluR5/6/7), and NMDA (NR1) ionotropic glutamate receptor subunits and to determine their distribution during the development of the chick retina by Western blotting and immunohistochemistry. Western blotting analysis at 1 day after hatching indicated that the antibodies against GluR1, 2/3, 4, and 5/6/7 and NR1 recognized specifically a single band of 100-110 kDa. In turn, immunohistochemistry at P1 showed that all subunits were expressed in cells of the inner nuclear and ganglion cell layers of the chick retina, mostly amacrine and ganglion cells, and their processes in the inner plexiform layer. In addition, stained processes in the outer plexiform layer were observed with the antibodies against GluR2/3, GluR4, and GluR5/6/7. Although all subunits appeared around E5-E6 in the prospective ganglion cell layer, and later in the prospective inner nuclear layer, the distribution of cells containing these glutamate receptor subunits revealed distinct ontogenetic patterns. This multiplicity of glutamate receptors may contribute to different processes that occur in the chick retina during development.
|Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb. |
Sassoè-Pognetto, M and Ottersen, O P
J. Neurosci., 20: 2192-201 (2000) 2000
Dendrodendritic synapses between mitral (or tufted) and granule cells of the olfactory bulb play a major role in the processes of odor discrimination and olfactory learning. Release of glutamate at these synapses activates postsynaptic receptors on the dendritic spines of granule cells, as well as presynaptic NMDA receptors in the mitral cell membrane. However, immunocytochemical studies have failed to demonstrate the presence of ionotropic glutamate receptors in granule cell dendrites. By using a postembedding immunogold procedure, we describe here the precise organization of neurotransmitter receptors at dendrodendritic synapses. We show that there is a selective localization of glutamate and GABA receptors at asymmetric and symmetric synaptic junctions, respectively. In addition, we demonstrate that NMDA and AMPA receptors are clustered at postsynaptic specializations on granule cell spines and that they are extensively colocalized. Conversely, glutamate receptors do not appear to be concentrated in clusters on mitral cell dendrites, suggesting that the presynaptic effects of glutamate are mediated by a small complement of extrasynaptic receptors. By analyzing the subsynaptic distribution of the NR1 and GluR2/3 subunits, we show that they are distributed along the entire extent of the postsynaptic specialization, indicating that both NMDA and AMPA receptors are available for dendrodendritic signaling between mitral and granule cells. These results indicate that the principles recently found to underlie the organization of glutamate receptors at axospinous synapses also apply to dendrodendritic synapses.
|EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. |
Dalva, M B, et al.
Cell, 103: 945-56 (2000) 2000
EphB receptor tyrosine kinases are enriched at synapses, suggesting that these receptors play a role in synapse formation or function. We find that EphrinB binding to EphB induces a direct interaction of EphB with NMDA-type glutamate receptors. This interaction occurs at the cell surface and is mediated by the extracellular regions of the two receptors, but does not require the kinase activity of EphB. The kinase activity of EphB may be important for subsequent steps in synapse formation, as perturbation of EphB tyrosine kinase activity affects the number of synaptic specializations that form in cultured neurons. These findings indicate that EphrinB activation of EphB promotes an association of EphB with NMDA receptors that may be critical for synapse development or function.
|Distribution of AMPA receptor subunits GluR1-4 in the dorsal vagal complex of the rat: a light and electron microscope immunocytochemical study. |
Kessler, J P and Baude, A
Synapse, 34: 55-67 (1999) 1999
The dorsal vagal complex, localized in the dorsomedial medulla, includes the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus nerve (DMN) and the area postrema (AP). The distribution of AMPA-preferring glutamate receptors (AMPA receptors) within this region was investigated using immunohistochemistry and antibodies recognizing either one (GluR1 or GluR4) or two (GluR2 and GluR3) AMPA receptors subunits. The distribution of GluR1 immunoreactivity showed high contrast of staining between strongly and lightly labeled areas. Labeling was intense in the AP and weak in the NTS, except for its medial and dorsalmost parts which exhibited moderate staining. Almost no GluR1 immunoreactivity was found in the DMN. GluR2/3 immunolabeling was present in the entire dorsal vagal complex. This labeling was strong in the AP, the DMN and the medial half of the NTS and moderate in the lateral half of the NTS, except for the interstitial subdivision which exhibited intense staining. Labeling induced by the GluR4 antibody was very weak throughout the dorsal vagal complex. Ultrastructural examination showed that GluR1 and GluR2/3 immunoreactivity was localized in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Immunoperoxidase staining in labeled cell bodies and dendrites was associated with intracellular organelles (microtubules, mitochondria, cisternae of the endoplasmic reticulum,.) and/or parts of the plasma membrane. Plasma membrane labeling was often associated with asymmetrical synaptic differentiations. No labeled symmetrical synapse was found using either GluR1 or GluR2/3 antibody. The present results show that AMPA receptors have a widespread distribution in neuronal perikarya and dendrites of the rat dorsal vagal complex. They suggest differences in subunit composition between AMPA receptors localized in the NTS, the DMN and the AP. Ultrastructural data are consistent with the fact that AMPA receptors associated with the plasma membrane are mostly synaptic receptors. However, they also suggest the existence of a large intracellular pool of receptor subunits in neuronal soma and dendrites.
|Differential expression of glutamate receptors by the dopaminergic neurons of the primate striatum. |
Betarbet, R and Greenamyre, J T
Exp. Neurol., 159: 401-8 (1999) 1999
Neostriatal neurons are targets of glutamatergic input from the cortex and thalamus. Glutamate receptors are abundantly, but differentially, expressed by the striatal neurons. We previously described the presence of dopaminergic cells intrinsic to the primate striatum that increase in number following MPTP treatment. In this study we have used double-label immunocytochemistry to analyze the expression of the glutamate receptor subunits GluR1, GluR2/3, NR1, mGluR1, and mGluR5 in the dopaminergic cells of the striatum. Our results show that 75% of these cells express GluR1 while 25% of them express NR1. They do not express GluR2/3 or the group 1 metabotropic receptors. Our results suggest that this potentially important population of cells expresses only calcium-permeable ionotropic glutamate receptors. We speculate that glutamate may play a role in regulating the number of these dopaminergic neurons after MPTP treatment and may also influence their ability to release dopamine.
|Localization of L-glutamate and glutamate-like receptors at the squid giant synapse. |
Di Cosmo, A, et al.
Brain Res., 839: 213-20 (1999) 1999
HPLC analysis of the amino acid contents of the second- and third-order giant fibres at the giant synapse in the stellate ganglion of the squid Loligo vulgaris shows that there are significantly higher amounts of L-glutamate and L-aspartate in the second-order (presynaptic) fibre than in the third-order (postsynaptic) fibre. Immunocytochemical staining of sections of the ganglion with an antibody raised against L-glutamate produces specific positive staining in the synaptic region of the second-order fibre. In contrast, staining with antibodies raised against glutamate-receptors (mammalian GluR1 with GluR2/3) produces positive staining in the third-order fibre at the postsynaptic region. These data provide further evidence for the hypothesis that L-glutamate is an excitatory transmitter at the giant synapse.
|Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat. |
García Del Caño, G, et al.
J. Neurocytol., 28: 455-68 (1999) 1999
The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca(2+)-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca(2+)-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.
|Hormonal regulation of glutamate receptor gene expression in the anteroventral periventricular nucleus of the hypothalamus. |
Gu, G, et al.
J. Neurosci., 19: 3213-22 (1999) 1999
Glutamate plays an important role in mediating the positive feedback effects of ovarian steroids on gonadotropin secretion, and the preoptic region of the hypothalamus is a likely site of action of glutamate. The anteroventral periventricular nucleus (AVPV) of the preoptic region is an essential part of neural pathways mediating hormonal feedback on gonadotropin secretion, and it appears to provide direct inputs to gonadotropin releasing hormone (GnRH)-containing neurons. Immunohistochemistry and in situ hybridization were used in this study to define the distribution and hormonal regulation of glutamate receptor subtypes in the AVPV of juvenile female rats. Neurons that express the NMDAR1 receptor subtype are abundant in the AVPV, as are cells that express AMPA receptor subtypes (GluR1, GluR2, and GluR3 but not GluR4), and the AVPV appears to contain a dense plexus of NMDAR1-immunoreactive presynaptic terminals. However, AVPV neurons do not seem to express detectable levels of kainate receptor (GluR5, GluR6, and GluR7) or metabotropic receptor (mGluR1-6) subtypes. Treatment of ovariectomized juvenile rats with estradiol induced expression of GluR1 mRNA but did not alter levels of GluR2 or GluR3 mRNA. Treatment of estrogen-primed ovariectomized juvenile rats with progesterone caused an initial increase in GluR1 mRNA expression, followed by a small decrease 24 hr after treatment. In contrast, estrogen appears to suppress levels of NMDAR1 mRNA in the AVPV, which remained unchanged after progesterone treatment. Thus, one mechanism whereby ovarian steroids may provide positive feedback to GnRH neurons is by altering the sensitivity of AVPV neurons to glutamatergic activation.
|Neuroprotective actions of peripherally administered insulin-like growth factor I in the injured olivo-cerebellar pathway. |
Fernandez, A M, et al.
Eur. J. Neurosci., 11: 2019-30 (1999) 1999
Exogenous administration of insulin-like growth factor I (IGF-I) restores motor function in rats with neurotoxin-induced cerebellar deafferentation. We first determined that endogenous IGFs are directly involved in the recovery process because infusion of an IGF-I receptor antagonist into the lateral ventricle blocks gradual recovery of limb coordination that spontaneously occurs after partial deafferentation of the olivo-cerebellar circuitry. We then analysed mechanisms whereby exogenous IGF-I restores motor function in rats with complete damage of the olivo-cerebellar pathway. Treatment with IGF-I normalized several markers of cell function in the cerebellum, including calbindin, glutamate receptor 1 (GluR1), gamma-aminobutyric acid (GABA) and glutamate, which are all depressed after 3-acetylpyridine (3AP)-induced deafferentation. IGF-I also promoted functional reinnervation of the cerebellar cortex by inferior olive (IO) axons. In the IO, increased expression of bax in neurons and bcl-X in astrocytes after 3AP was significantly reduced by IGF-I treatment. On the contrary, IGF-I prevented the decrease in poly-sialic-acid neural cell adhesion molecule (PSA-NCAM) and GAP-43 expression induced by 3AP in IO cells. IGF-I also significantly increased the number of neurons expressing bcl-2 in brainstem areas surrounding the IO. Altogether, these results indicate that subcutaneous IGF-I therapy promotes functional recovery of the olivo-cerebellar pathway by acting at two sites within this circuitry: (i) by modulating death- and plasticity-related proteins in IO neurons; and (ii) by impinging on homeostatic mechanisms leading to normalization of cell function in the cerebellum. These results provide insight into the neuroprotective actions of IGF-I and may be of practical consequence in the design of new therapeutic approaches for neurodegenerative diseases.
|Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning. |
Zamanillo, D, et al.
Science, 284: 1805-11 (1999) 1999
Gene-targeted mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR-A exhibited normal development, life expectancy, and fine structure of neuronal dendrites and synapses. In hippocampal CA1 pyramidal neurons, GluR-A-/- mice showed a reduction in functional AMPA receptors, with the remaining receptors preferentially targeted to synapses. Thus, the CA1 soma-patch currents were strongly reduced, but glutamatergic synaptic currents were unaltered; and evoked dendritic and spinous Ca2+ transients, Ca2+-dependent gene activation, and hippocampal field potentials were as in the wild type. In adult GluR-A-/- mice, associative long-term potentiation (LTP) was absent in CA3 to CA1 synapses, but spatial learning in the water maze was not impaired. The results suggest that CA1 hippocampal LTP is controlled by the number or subunit composition of AMPA receptors and show a dichotomy between LTP in CA1 and acquisition of spatial memory.
|Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. |
Shi, S H, et al.
Science, 284: 1811-6 (1999) 1999
To monitor changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events required synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediatedtransmission observed during long-term potentiation and activity-dependent synaptic maturation.
|A diffusion barrier maintains distribution of membrane proteins in polarized neurons. |
B Winckler, P Forscher, I Mellman
Nature 397 698-701 1999
The asymmetric distribution of proteins to distinct domains in the plasma membrane is crucial to the function of many polarized cells. In epithelia, distinct apical and basolateral surfaces are maintained by tight junctions that prevent diffusion of proteins and lipids between the two domains. Polarized neurons maintain axonal and somatodendritic plasma membrane domains without an obvious physical barrier. Indeed, the artificial lipid Dil encounters no diffusion barrier at the presumptive domain boundary, the axon hillock. By measuring the lateral mobility of membrane proteins using optical tweezers, we show here that some membrane proteins exhibit markedly reduced mobility in the initial segment of the axon. Disruption of F-actin and low levels of dimethyl sulphoxide (DMSO) abolish this diffusion barrier and lead to redistribution of membrane markers that had previously been polarized. Immobilization in the initial segment may reflect, at least in part, differential tethering to cytoskeletal components. Therefore, the ability to maintain a polarized distribution of membrane proteins depends on a specialized domain at the initial segment of the axon, which restricts lateral mobility and serves as a new type of diffusion barrier that acts in the absence of cell-cell contact.
|An alternative pathway for rod signals in the rodent retina: rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors. |
Hack, I, et al.
Proc. Natl. Acad. Sci. U.S.A., 96: 14130-5 (1999) 1999
In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.
|Dual (excitatory and inhibitory) calretinin innervation of AMPA receptor-containing neurons in the rat lateral septum. |
Holderith, N, et al.
Experimental brain research. Experimentelle Hirnforschung. Expérimentation cérébrale, 119: 65-72 (1998) 1998
A recent study demonstrated both an extrinsic and an intrinsic calretinin (CR) innervation of the rat septal complex and that a population of the extrinsic calretinin fibers is aspartate/glutamate-containing. The aim of this study was to determine which types (GluR1, GluR2/3, or both) of AMPA receptor-containing lateral septal area neurons are innervated by extrinsic and intrinsic CR neurons and whether the intrinsic CR cells are GABAergic. Light- and electron-microscopic single immunostaining for CR, GluR1, and GluR2/3, as well as light- and electron-microscopic-double immunostaining experiments for CR plus GluR1 and CR plus GluR2/3 were performed in the lateral septal area. Furthermore, the "mirror" colocalization technique was employed on consecutive vibratome sections of the septal complex to investigate whether the intrinsic septal CR neurons are GABAergic. The results are summarized as follows: (1) both GluR1- and GluR2/3-immunoreactive neurons are innervated by CR-containing fibers; (2) the majority of these synapses, observed mainly on the soma and, to a lesser extent, on proximal dendrites of AMPA receptor-containing neurons, represent asymmetric synaptic membrane specializations; (3) a minority of CR-containing axon terminals associated with both GluR1- and GluR2/3-immunoreactive neurons form symmetric contacts, predominantly on their soma; and (4) 93% of the lateral septal area CR cells are GABAergic. These observations indicate that both GluR1- and GluR2/3-containing lateral septal area neurons receive a dual intrinsic and extrinsic CR innervation. The former (intrinsic) CR boutons are GABAergic, while the latter form asymmetric synaptic contacts, are excitatory, and probably originate in the supramammillary area, since previous work has demonstrated that a population of supramammillo-septal fibers contain aspartate and/or glutamate.
|Light-induced retinal degeneration suppresses developmental progression of flip-to-flop alternative splicing in GluR1. |
Harada, T, et al.
J. Neurosci., 18: 3336-43 (1998) 1998
AMPA receptors are hetero-oligomers composed of subsets of four distinct subunits, termed GluR1, GluR2, GluR3, and GluR4. Using quantitative reverse transcription-PCR analysis, we have found that light-induced degeneration of rat retina dramatically suppresses developmental progression of the flip-to-flop alternative splicing switch of retinal GluR1 mRNA. When animals were raised under standard conditions of a 12 hr light/dark cycle (LD 12:12), the flop-to-flip ratio in GluR1 and GluR2 dramatically increased between postnatal day 10 (P10) and P28, and the ratios continued to increase gradually up to P84. When animals were raised in complete darkness, this increase was delayed in GluR1 between P21 and P42. In addition, the increase of the flop-to-flip ratio in GluR1 was strongly suppressed after P21 under conditions of continuous illumination from P2. This is significant because P21 is just after the eye opening and is the timing of the onset of light-induced retinal degeneration. This suppression of the increase of the flop-to-flip ratio was specific to GluR1 and was not observed in GluR2-4. Immunocytochemistry and immunoblot analysis suggested no changes in either the distribution or expression of GluR1 protein in the light-damaged retina measured at P84. When rats were raised under continuous illumination from P2 to P21 followed by LD 12:12 from P22 to P84, retinal degeneration did not progress after P22. In such animals the flop-to-flip ratio, once decreased to approximately 50% of the control (LD 12:12) at P21, was restored to the control level at P84. These findings demonstrate that developmental progression of the flip-to-flop exon switch in retinal GluR1 is affected by lighting conditions, and that light-induced retinal degeneration contributes to the mechanism of suppression of this splicing switch.
|The postnatal development of AMPA receptor subunits in the basal ganglia of the rat. |
Jakowec, M W, et al.
Dev. Neurosci., 20: 19-33 (1998) 1998
In situ hybridization and immunohistochemistry were used to characterize the expression pattern of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors during postnatal development in the rat basal ganglia. All subunit transcripts showed some degree of developmental regulation. GluR1 and GluR2 are expressed at high levels in the neonate with reduced expression in the adult. GluR3 and GluR4 are expressed at significantly lower levels in both neonates and adults and have much more modest degrees of reduced expression in adults as compared with GluR1 and GluR2. Analysis of the flip and flop transcript isoforms indicates that GluR1 flip and flop and GluR2 flip are the predominately expressed splice variants in adults. Observed changes in the expression of the AMPA receptor transcripts indicate that there are fundamental differences in the expression of these receptor subunits in adults and neonates. This phenomenon may play a significant role in the establishment of proper synaptic circuitry within the developing basal ganglia in early postnatal life as well as contributing to differences in susceptibility to injury and disease in the aging brain.
|Neurochemistry of the mammalian cone 'synaptic complex'. |
Vardi, N, et al.
Vision Res., 38: 1359-69 (1998) 1998
The cone 'synaptic complex' is a unique structure in which a single presynaptic axon secretes glutamate onto processes of bipolar cells (both ON and OFF) and horizontal cells. In turn, the horizontal cell processes antagonize cone and bipolar responses to glutamate (probably by GABA). What still remains largely unknown is the molecular identity of the postsynaptic receptors and their exact locations. We identified several subunits of the glutamate receptor and the GABAA receptor expressed at the cone synaptic complex and localized them ultrastructurally. Glutamate receptors: (i) Invaginating (probably ON) bipolar dendrites in the monkey and rat express the metabotropic glutamate receptor, mGluR6. The stain is intense on the dendritic membrane where it first enters the invagination, and weak at the tip nearest to the ribbon. The cone membrane is electron-dense where it apposes the intense stain for mGluR6. Surprisingly, invaginating bipolar dendrites in the cat also express the AMPA receptor subunits, GluR2/3 and GluR4. (ii) Dendrites forming basal contacts in the cat (probably OFF) express the AMPA subunits GluR2/3, GluR4, and also the kainate subunit, GluR6/7. The stain is especially intense at the dendritic tips in apposition to electron-dense regions of cone membrane. (iii) Horizontal cells in the cat express the AMPA subunits GluR2/3, GluR4 and the kainate subunit, GluR6/7. The stain is strongest in the cytosol of somas and primary dendrites, but is also present in the invaginating terminals where it localizes to the membrane subjacent to the ribbon. GABAA receptors: (i) ON and OFF bipolar dendrites in the monkey express the alpha 1 and beta 2/3 subunits. The stain is localized to the bipolar cell membrane in apposition to horizontal cell processes. (ii) Cones did not express the GABAA subunits tested by immunocytochemistry, but beta 3 mRNA was amplified by RT-PCR from rat photoreceptors. Conclusions: (i) mGluR6 receptors concentrate on dendrites at the base of the invagination rather than at the apex. This implies that receptors at both 'invaginating' and 'basal' contacts lie roughly equidistant from the release sites and should therefore receive similar spatiotemporal concentrations of glutamate. (ii) The 'cone' membrane is electron-dense opposite to the receptor sites on both ON and OFF bipolar cells. This suggests a special role for this region in synaptic transmission. Possibly, these densities signify a transporter that would regulate glutamate concentration at sites remote (> 200 nm) from the locus of vesicle release.
|Maintenance of late-phase LTP is accompanied by PKA-dependent increase in AMPA receptor synthesis. |
Nayak, A, et al.
Nature, 394: 680-3 (1998) 1998
Long-term potentiation (LTP) is a form of synaptic plasticity that has been extensively studied as a putative mechanism underlying learning and memory. A late phase of LTP occurring 3-5 hours after stimulation and depending on transcription, protein synthesis and cyclic-AMP-dependent protein kinase (protein kinase A, or PKA) has been described, but it is not known whether transcription of presynaptic and/or postsynaptic genes is required to support late-phase LTP. Here we show that late-phase LTP can be obtained in rat hippocampal CA1 mini-slices in which the cell bodies of presynaptic Schaffer collateral/commissural fibres are removed. Thus, transcription of presynaptic genes is not necessary to support maintenance of late-phase LTP. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor is the predominant mediator of the ionotropic response to synaptically released glutamate in the hippocampus and it has been implicated in LTP maintenance. We find that synthesis of AMPA receptor subunits is increased three hours after LTP induction: this effect on the synthesis of the AMPA receptor is blocked by inhibitors of PKA and of transcription. Our results support the idea of a postsynaptic mechanism maintaining late-phase LTP, in which AMPA receptor synthesis is increased as a result of PKA-dependent gene transcription.
|GluR1 and GluR2/3 subunits of the AMPA-type glutamate receptor are associated with particular types of neurone in laminae I-III of the spinal dorsal horn of the rat. |
Kerr, R C, et al.
Eur. J. Neurosci., 10: 324-33 (1998) 1998
GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor are expressed at high levels by neurones in laminae I-III of rat spinal dorsal horn, an area which contains numerous, densely packed small neurones. In order to determine whether these subunits are expressed by inhibitory or excitatory neurones, we combined pre-embedding immunocytochemistry with antibodies that recognize either GluR1, or an epitope common to GluR2 and 3, with postembedding detection of gamma-aminobutyric acid (GABA) and glycine. Most (78%) of the neurones with GluR1-immunoreactivity were GABA-immunoreactive, and some of these were also glycine-immunoreactive, whereas nearly all (97%) of the GluR2/3-immunoreactive neurones were not GABA- or glycine-immunoreactive. We carried out double-immunofluorescence and confocal microscopy to provide further information on the neurochemistry of cells that express these subunits. As expected, all neurotensin- and virtually all somatostatin-immunoreactive cells (which are thought to be excitatory interneurones) were GluR2/3- but not GluR1-immunoreactive, whereas parvalbumin-containing cells (most of which are GABAergic) possessed GluR1-, but usually not GluR2/3-immunoreactivity. Neurones that contained nitric oxide synthase (most of which are GABAergic) were more variable, with 57% GluR1-immunoreactive and 41% GluR2/3-immunoreactive. Cholinergic neurones in lamina III (which are also GABAergic) invariably showed each type of GluR-immunoreactivity. These results suggest that neuronal populations in laminae I-III have characteristic patterns of GluR expression: GluR1 is particularly associated with inhibitory neurones, and GluR2 with excitatory neurones. This makes it likely that some of the AMPA receptors present on the inhibitory interneurones lack the GluR2 subunit, and may therefore have significant Ca2+-permeability.
|Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway. |
Baranes, D, et al.
Neuron, 21: 813-25 (1998) 1998
The expression of tissue plasminogen activator (tPA) is increased during activity-dependent forms of synaptic plasticity. We have found that inhibitors of tPA inhibit the late phase of long-term potentiation (L-LTP) induced by either forskolin or tetanic stimulation in the hippocampal mossy fiber and Schaffer collateral pathways. Moreover, application of tPA enhances L-LTP induced by a single tetanus. Exposure of granule cells in culture to forskolin results in secretion of tPA, elongation of mossy fiber axons, and formation of new, active presynaptic varicosities contiguous to dendritic clusters of the glutamate receptor R1. These structural changes are blocked by tPA inhibitors and induced by application of tPA. Thus, tPA may be critically involved in the production of L-LTP and specifically in synaptic growth.
|Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy. |
Z Ying, T L Babb, Y G Comair, M Bushey, K Touhalisky
Brain research 798 239-46 1998
In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of 'excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P0.000001) and GluR1 protein densities were significantly higher (P0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.
|Kainate/AMPA receptors expressed on human fetal astrocytes in long-term culture. |
Cauley, K, et al.
J. Neurosci. Res., 47: 311-21 (1997) 1997
Long-term cultivation of primary human fetal brain cells has yielded a homogeneous population of glial progenitors of extended life span. These human astrocyte precursor (HAP-1) cells have been in culture for greater than 1 year, are diploid, and do not form colonies in soft agar. The culture was established in 10% fetal calf serum (FCS), although cells greatly increase their proliferative rate when both basic fibroblast growth factor and FCS are present in the culture media. HAP-1 cells express the cytoskeletal proteins glial fibrillary acidic protein, vimentin, and nestin. HAP-1 cells express the AMPA/kainate receptor subunit genes GluRs 1, 3, and 4 and the kainate receptor subunit genes GluR6, KA1, and KA2. Immunohistochemistry confirms the expression of GluR subunit proteins. HAP-1 cells demonstrate a kainate-responsive current found to be blockable by CNQX. HAP-1 cells will serve in the study of human glial cells and ligand-gated ion channels and in the identification of compounds which might act as agonists or antagonists at these receptor-ion channel complexes.
|Glutamate receptors in the olfactory bulb synaptic circuitry: heterogeneity and synaptic localization of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1. |
Giustetto, M, et al.
Neuroscience, 76: 787-98 (1997) 1997
In this study, we analysed the molecular heterogeneity and synaptic localization of the N-methyl-D-aspartate receptor subunit 1 and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit 1 in the olfactory bulb glomerular synaptic circuitry. Semiquantitative reverse transcriptase polymerase chain reaction showed that approximately 40% of the N-methyl-D-aspartate receptor subunit 1 messenger RNA splice variants contain the N1 exon, which conveys specific functional properties on the channel. In other forebrain and hindbrain regions that we examined, the ratio of the N1-containing (receptor subunit 1(1XX)) to N1-lacking (receptor subunit 1(0XX)) N-methyl-D-aspartate receptor subunit 1 messenger RNAs varied considerably. The cellular and subcellular distribution of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 was investigated with antibodies generated against the C-terminal domain of the individual subunits [Petralia R. S. et al. (1994) J. Neurosci. 14, 667 696; Wenthold R. J. et al. (1992) J. biol Chem. 267, 501 507]. Both N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 were localized to the postsynaptic density of asymmetric synapses established by olfactory receptor neuron terminals with the dendrites of mitral and tufted cells. Not all of these synapses, however, were labelled. These results are consistent with the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral and tufted cell synapses, and suggest a high heterogeneity in the expression of the postsynaptic glutamate receptors.
|Translocation of autophosphorylated calcium/calmodulin-dependent protein kinase II to the postsynaptic density. |
Strack, S, et al.
J. Biol. Chem., 272: 13467-70 (1997) 1997
Calcium/calmodulin-dependent protein kinase II (CaMKII) undergoes calcium-dependent autophosphorylation, generating a calcium-independent form that may serve as a molecular substrate for memory. Here we show that calcium-independent CaMKII specifically binds to isolated postsynaptic densities (PSDs), leading to enhanced phosphorylation of many PSD proteins including the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-type glutamate receptor. Furthermore, binding to PSDs changes CaMKII from a substrate for protein phosphatase 2A to a protein phosphatase 1 substrate. Translocation of CaMKII to PSDs occurs in hippocampal slices following treatments that induce CaMKII autophosphorylation and a form of long term potentiation. Thus, synaptic activation leads to accumulation of autophosphorylated, activated CaMKII in the PSD. This increases substrate phosphorylation and affects regulation of the kinase by protein phosphatases, which may contribute to enhancement of synaptic strength.
|Calcium- and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the post-synaptic density. |
Hayashi, Y, et al.
Brain Res. Mol. Brain Res., 46: 338-42 (1997) 1997
We have detected immunoreactivities of AMPA receptor subunits GluR1-4 in post-synaptic density (PSD) fraction and tested whether they can be phosphorylated by endogenous kinases. Incubation of PSD with Ca2+ and calmodulin increased phosphorylation of GluR1 and GluR2/3. The phosphorylation of GluR1 was largely blocked by a Ca2+/calmodulin-dependent protein kinase type II inhibitor. Thus Ca2+/calmodulin-dependent phosphorylation of glutamate receptor may be a mechanism underlying enhanced post-synaptic receptor responsiveness in LTP.
|Sensitization to morphine induced by viral-mediated gene transfer. |
Carlezon, W A, et al.
Science, 277: 812-4 (1997) 1997
Repeated administration of morphine sensitizes animals to the stimulant and rewarding properties of the drug. It also selectively increases expression of GluR1 (an AMPA glutamate receptor subunit) in the ventral tegmental area, a midbrain region implicated in morphine action. By viral-mediated gene transfer, a causal relation is shown between these behavioral and biochemical adaptations: Morphine's stimulant and rewarding properties are intensified after microinjections of a viral vector expressing GluR1 into the ventral tegmental area. These results confirm the importance of AMPA receptors in morphine action and demonstrate specific locomotor and motivational adaptations resulting from altered expression of a single localized gene product.
|GABAergic neurons with AMPA GluR1 and GluR2/3 immunoreactivity in the rat striate cortex. |
Gutiérrez-Ibarluzea, I, et al.
Neuroreport, 8: 2495-9 (1997) 1997
The co-localization of GABA with AMPA receptor subunits GluR1 or GluR2/3 was analysed in the striate cortex of adult rats by post-embedding immunocytochemistry in semithin sections. Adjacent 1 micron semithin sections of four brains were alternately incubated with specific antibodies against GABA and the GluR1 and GluR2/3 subunits. The post-embedding immunocytochemistry showed that 38% of GABAergic neurons contained the GluR1 subunit and 10% contained the GluR2/3 subunits. Previous work has shown GluR1 immunoreactivity in non-pyramidal neurons and GluR2/3 immunoreactivity in pyramidal neurons. However, this study is the first to demonstrate that there are GABAergic neurons co-localized with GluR2/3 AMPA subunits. Additionally, this study provides quantitative estimations of the laminar distribution of GABAergic and non-GABAergic cells containing the AMPA GluR1 and GluR2/3 subunits.
|Expression of glutamate receptor subunit 1 and nitric oxide synthase in the hypoglossal nucleus and dorsal vagal nucleus in the rat after neurectomy. |
Yeo, J F, et al.
Int. J. Neurosci., 90: 9-20 (1997) 1997
Using nitric oxide synthase (NOS) and glutamate receptor subunit 1 (GluR1) immunohistochemistry, the present study demonstrated changes in the expression of NOS and GluR1 in the hypoglossal (HN) and dorsal vagal nucleus (DVN) after neurectomy. Two and 7 days after sectioning the left hypoglossal nerve, NOS expression was seen in a few neurons but GluR1 immunoreactivity was drastically reduced in the ipsilateral HN. The upregulation of NOS immunoreactivity in the HN appeared to peak at 14 days postoperation (dpo). At this period, however, the GluR1 immunoreactivity almost completely disappeared. Twenty-one, 35 and 56 days after neurectomy, NOS immunoreactivity was still expressed in the ipsilateral HN; at the same time, GluR1 immunoreactivity reappeared in a few neurons of the nucleus. Ninety days after operation, NOS immunoreactivity completely disappeared on the operated side of the nucleus, but GluR1 immunoreactivity was re-expressed in many hypoglossal neurons. The number of such neurons was obviously less than that on the unoperated side. After sectioning the left vagus nerve in the same animals, the expression of NOS immunoreactivity in the ipsilateral DVN resembled that in the HN. On the unoperated side, NOS immunoreactivity was demonstrated in some neurons in the DVN, like that in the normal. In both normal and operated rats, only a few neurons expressed GluR1 immunoreactive products on both the operated and unoperated sides of the DVN. Combining with previous results on protein synthesis observed at 14 dpo, the present investigation suggested that in the early stages after neurectomy, the expression of NOS immunoreactivity and loss of GluR1 expression in the HN may indicate the organism's double protective mechanism. Lastly, the reappearance of GluR1 in the same nucleus from 21 to 90 days after operation may reflect functional recovery of the hypoglossal neurons.
|Modulation of AMPA receptor subunits GluR1 and GluR2/3 in the rat cerebellum in an experimental hepatic encephalopathy model. |
Suárez, I, et al.
Brain Res., 778: 346-53 (1997) 1997
The immunohistochemical expression and distribution of the AMPA-selective receptor subunits GluR1 and GluR2/3 were investigated in the rat cerebellum following portocaval anastomosis (PCA) at 1 and 6 months. With respect to controls, GluR1 and GluR2/3 immunoreactivities increased over 1 to 6 months following PCA, although immunolabelling patterns for both antibodies were different at the two analysed times. GluR1 immunoreactivity was expressed by Bergmann glial cells, which showed immunoreactive glial processes crossing the molecular layer at 6 months following PCA. The GluR2/3 subunit was expressed by Purkinje neurons and moderately expressed by neurons of the granule cell layer. Immunoreactivity for GluR2/3 was detectable in cell bodies and dendrites of Purkinje cells in young control cerebella, whereas GluR2/3 immunoreactivity was scarce 1 month post PCA. However, despite a lack of immunoreactivity in the Purkinje somata and main processes of adult control rats, GluR2/3 immunoreactivity was strongly enhanced in Purkinje neurons following long-term PCA. These findings suggest that the localization of the GluR2/3 subunit in Purkinje cells undergoes an alteration and/or reorganization as a consequence of long-term PCA. The combination of enhanced GluR immunoreactivity in long-term PCA, both in Bergmann glial cells and in Purkinje neurons, suggests some degree of neuro-glial interaction, possibly through glutamate receptors, in this type of encephalopathy.
|Cellular, subcellular, and subsynaptic distribution of AMPA-type glutamate receptor subunits in the neostriatum of the rat. |
V Bernard, P Somogyi, J P Bolam
The Journal of neuroscience : the official journal of the Society for Neuroscience 17 819-33 1997
Glutamate released in the basal ganglia is involved in the expression of clinical symptoms of neurodegenerative diseases like Parkinson's or Huntington's. Neostriatal neurons are the targets of glutamatergic inputs derived from the cortex and the thalamus acting via AMPA-type as well as other glutamate receptors. To determine the location of subunits of the AMPA subclass of glutamate receptors (GluR) in the rat neostriatum, we applied multiple immunocytochemical techniques using anti-peptide antibodies against the GluR1, GluR2/3, and GluR4 subunits at both the light and electron microscopic levels. All medium spiny efferent neurons, some of which were identified as striatonigral neurons, displayed immunoreactivity for GluR1 and GluR2/3 subunits. Double immunofluorescence revealed that at least 70-90% of parvalbumin-immunopositive GABAergic interneurons were immunoreactive for each of GluR1, GluR2/3, or GluR4 subunits and that at least 40% of choline acetyltransferase-immunopositive cholinergic interneurons were immunopositive for GluR1 or GluR4 subunits. The majority of nitric oxide synthase-immunopositive neurons had no detectable immunoreactivity for any of the AMPA receptor subunits. Electron microscopic analysis confirmed the presence of immunoreactivity for GluR1 and GluR2/3 in the perikarya of spiny neurons and interneurons and GluR4 in perikarya of interneurons only. GluR1 and GluR2/3 subunits were detected in dendrites and spines. A significant population of extrasynaptic receptors was revealed by pre-embedding immunogold labeling along the plasma membranes of perikarya, dendrites, and spines. Receptors were concentrated in the postsynaptic membrane specialization of asymmetrical synapses, as revealed by the postembedding immunogold method. Quantitative analysis demonstrated that immunoreactivity for the GluR1 and GluR2/3 subunits is higher at the periphery than at the middle of the postsynaptic membrane specialization. Our results demonstrate that AMPA receptor subunits are distributed widely and heterogeneously among striatal neurons and are concentrated on the postsynaptic membrane of asymmetrical synaptic specializations, although extrasynaptic receptors are also present.
|Expression of ionotropic glutamate receptor subunits in glial cells of the hippocampal CA1 area following transient forebrain ischemia. |
Gottlieb, M and Matute, C
J. Cereb. Blood Flow Metab., 17: 290-300 (1997) 1997
We examined by immunohistochemistry the expression of ionotropic glutamate receptor subunits (GluRs) in glial cells of the rat dorsal hippocampus 3 to 28 days after transient forebrain ischemia. In general, the expression of GluRs at all time points studied underwent a drastic reduction that was primarily restricted to the CA1 region. In addition to the disappearance of GluRs as a result of neuronal cell death, we observed their expression in reactive glial cells. The time course of expression and the subunits involved were different for astrocytes and microglia. Reactive astrocytes exhibited kainate, GluR5-7, and N-methyl-D-aspartate (NMDA), NR2A/B, receptor subunits, both of which were maximally expressed approximately 4 weeks after ischemia. In contrast, reactive microglia expressed GluR4 and NR1 subunits, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and NMDA receptor subtypes, respectively, with maximal expression observed between 3 and 7 days after ischemia. These results demonstrate that specific types of GluRs are expressed in reactive glial cells after ischemia and that, overall, their expression levels peak around or after the periods of maximal astrogliosis and microgliosis. Thus, modulation of GluR expression may be one of the molecular components accompanying the gliotic process.
|Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes. |
Matute, C, et al.
Proc. Natl. Acad. Sci. U.S.A., 94: 8830-5 (1997) 1997
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 alpha-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.
|Adenovirus-mediated expression of AMPA-type glutamate receptor channels in PC12 cells. |
M Sudo, K Tsuzuki, H Okado, A Miwa, S Ozawa
Brain research. Molecular brain research 50 91-9 1997
The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor channels are expressed ubiquitously in brain neurons and mediate fast excitatory neurotransmission. They are composed of four subunits, GluR1, GluR2, GluR3 and/or GluR4. We constructed recombinant adenoviruses encoding rat AMPA receptor subunit cDNAs, GluR1 (AxCAGluR1) and silently mutated GluR2 (AxCAGluR2X) with modified chicken beta-actin promoter and cytomegalovirus immediate-early enhancer. Using these adenoviral vectors, we transferred the GluR1 and GluR2 genes into PC12 cells that possess no functional AMPA receptor channels. PC12 cells infected with these viruses expressed GluR1 and GluR2 RNAs. Immunoblot analysis indicated that the expressed GluR1 and GluR2 proteins were equivalent to those of the rat brain. Functional expression of the AMPA receptor channels was examined using the whole-cell patch clamp technique. In AxCAGluR1-infected cells, the current-voltage (I-V) relationship of response to kainate, a non-desensitizing agonist of AMPA receptors, exhibited a strong inward rectification, indicating the formation of functional GluR1-homomeric channels. In cells infected with both AxCAGluR1 and AxCAGluR2X, the I-V relationship of kainate responses exhibited an outward rectification, indicating the formation of heteromeric GluR1/R2 channels. Immunocytochemical analysis revealed that the AMPA receptor subunit genes were transferred in more than 95% of the infected PC12 cells.
|Localization of AMPA glutamate receptor subunits in subpopulations of non-pyramidal neurons in the rat basolateral amygdala. |
McDonald, A J
Neurosci. Lett., 208: 175-8 (1996) 1996
A two-color immunoperoxidase technique was used to investigate the localization of GluR1 and GluR2/3 alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) glutamate receptor subunits in subpopulations of non-pyramidal neurons in the rat basolateral amygdala. Antibodies to neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and calcium binding proteins were used to mark three distinct subsets of non-pyramidal cells. Only non-pyramidal neurons containing the calcium binding proteins parvalbumin (PV) and calbindin D-28k (CB) exhibited robust GluR1-like immunoreactivity (GluR1-ir). Light GluR1-ir was observed in some neurons with NPY-ir and VIP-ir, as well as in some pyramidal cells. Only pyramidal cells exhibited robust GluR2/3-ir. No significant amount of GluR2/3-ir was seen in any of the three main classes of non-pyramidal cells. These findings indicate that in the rat basolateral amygdala GluR2/3-ir is found primarily in pyramidal neurons whereas GluR1-ir is found mainly in non-pyramidal neurons that contain PV or CB.
|Distribution of NMDA and AMPA receptors in the cerebellar cortex of rhesus macaques. |
Garyfallou, V T, et al.
Brain Res., 716: 22-8 (1996) 1996
The distribution of glutamate receptors in the cerebellar cortex of the rhesus macaque was examined by light microscopic immunocytochemistry using an antibody specific to the N-methyl-D-aspartate (NMDA) R1 receptor subunit (i.e. NMDAR1) as well as antibodies specific to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits (i.e. GluR1, GluR2/3, and GluR4). NMDAR1 immunolabeling was most prevalent in the Purkinje cell perikarya and dendrities, but was also significant in the stellate and basket cells of the granular layer and Golgi cells of the molecular layer. On the other hand, GluRl and GluR4 immunolabeling was concentrated principally in the processes of the Bergmann glia located in the vicinity of the Purkinje cell perikarya. Although GluR2/3 immunolabeling also occurred in these Bergmann glia processes as well as in the Bergmann fibers, it was more pronounced in the Purkinje cell perikarya and dendrites; additionally, significant GluR2/3 labeling was evident in the stellate and basket cells of the molecular layer and medium-size soma of the granular layer (most likely Golgi cells). In situ hybridization histochemistry (ISHH), using cRNA probes to NMDAR1. GluR1.GluR2, and GluR3, showed glutamate receptor mRNA distribution patterns consistent with those disclosed in the immunocytochemical study. Furthermore, the ISHH findings suggest that the positive immunocytochemical labeling of Purkinje cells with the GluR2/3 antibody is most likely due to the gene expression of both GluR2 and GluR3 AMPA receptor subtypes. Taken together, the results are potentially important for the elucidation of mechanisms that control aspects of cerebellar function, such as long-term depression.
|Ultrastructural localization of glutamate receptor subunits (NMDAR1, AMPA GluR1 and GluR2/3) and spinothalamic tract cells. |
Ye, Z and Westlund, K N
Neuroreport, 7: 2581-5 (1996) 1996
The associations of glutamate receptor subunits (NMDAR1, AMPA GluR1 and GluR2/3) and spinothalamic tract neurons in the rat lumbar spinal cord dorsal horn were investigated. Staining for NMDAR1 and AMPA GluR1 and GluR2/3 receptor subunits was observed throughout the spinothalamic tract soma and dendrites, particularly in association with the rough endoplasmic reticulum and some postsynaptic membrane sites. Immunostaining for NMDAR1 and AMPA GluR2/3 was also noted in presynaptic membrane sites. Localization of both NMDA and AMPA glutamate receptor subunits in association with spinothalamic tract neurons provides anatomical evidence in support of the various interactions reported for glutamate receptors in nociception. Presynaptic localization of the AMPA GluR2/3 receptor subunit suggests that spinothalamic tract cells may also be affected presynaptically by AMPA glutamate receptor interactions.
|Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons. |
Wenthold, R J, et al.
J. Neurosci., 16: 1982-9 (1996) 1996
The AMPA receptor, which is involved in most fast glutamatergic transmission in the mammalian brain and is expressed in most neurons, is made up of four subunits, GluR1-4. In situ hybridzation, immunocytochemistry studies, and single-cell PCR analyses show that the number and type of AMPA receptor subunits expressed vary among neuronal populations and that two to four subunits usually are expressed in each neuron. Neurons that express two or more subunits theoretically could produce multiple pentameric receptor complexes that differ in their subunit compositions, and these complexes could be targeted to different synaptic populations. To determine whether a single neuronal population produces multiple AMPA receptor complexes, we used a preparation of CA1/CA2 hippocampal pyramidal neurons and immunoprecipitation with subunit-specific antibodies to characterize the receptor complexes. The CA1/CA2 pyramidal neurons express high levels of GluR1-3 and receive multiple excitatory inputs, offering the possibility that distinct receptor complexes may be assembled and expressed selectively at different synaptic populations. Our results suggest the presence of two major populations of AMPA receptor complexes: those made up of GluR1 and GluR2 and those made up of GluR2 and GluR3. Very few complexes contained both GluR1 and GluR3, whereas approximately 8% of the total AMPA receptor complexes was homomeric GluR1. The integrity of the receptor complex was verified by measuring [3H]AMPA binding activity in the immunoprecipitated fractions. These results show that AMPA receptor complexes with different subunit compositions are present in CA1/CA2 pyramidal neurons and suggest an additional mechanism to regulate receptor expression in neurons.
|Expression of glutamate receptors in the human and rat basal ganglia: effect of the dopaminergic denervation on AMPA receptor gene expression in the striatopallidal complex in Parkinson's disease and rat with 6-OHDA lesion. |
Bernard, V, et al.
J. Comp. Neurol., 368: 553-68 (1996) 1996
The overactivity of subthalamopallidal and corticostriatal glutamatergic neurons observed in Parkinson's disease (PD) suggests that antagonists of glutamate receptor could be used to alleviate the motor symptoms of the disease. In this study, we analysed two features of the striatopallidal complex: (1) the distribution of alpha-amino-3 hydroxy-5-methyl-4-isoxasol-propionate (AMPA) and kainate receptors and their corresponding mRNA by immunohistochemistry and in situ hybridisation and (2) the effect of dopaminergic denervation on AMPA receptor gene expression in PD patients and rats with 6-hydroxydopamine (6-OHDA)-induced degeneration of the nigrostriatal dopaminergic system. All AMPA receptor mRNAs and proteins (GluR1-4) were detected in the internal segment of the globus pallidus (GPi). Among kainate receptors, only KA1 and KA2 were detectable and only at a low level. Only GluR4 protein was detected in the neuropil of the GPi. In the striatum, GluR1, GluR2, and GluR3 were detected in about 70% of medium-sized and large neurons. By contrast, GluR4 mRNA was detected in only a small number of large and medium-sized neurons. Among kainate receptors, GluR6, GluR7, and KA2 were detected in about 50-60% of medium-sized neurons, whereas GluR5 and KA1 were restricted to 1-2% and 20-30% of these neurons, respectively. These results suggest that antagonists of AMPA and kainate receptors could be effective in alleviating motor symptoms in Parkinson's disease by blocking the overstimulation of pallidal and striatal neurons by glutamate. A significant decrease in GluR1 gene expression (-33%) was observed in the neurons of the GPi in PD patients and in rat entopeduncular nucleus ipsilateral to the 6-OHDA lesion (-20%). GluR2, GluR3, and GluR4 mRNA levels in the GPi and GluR1-4 levels in the striatum were unchanged in PD patients and 6-OHDA-lesioned rats compared with their respective controls. These data suggest that dopamine positively regulates only GluR1 gene expression in the GPi.
|Cellular expression of ionotropic glutamate receptor subunits on specific striatal neuron types and its implication for striatal vulnerability in glutamate receptor-mediated excitotoxicity. |
Chen, Q, et al.
Neuroscience, 73: 715-31 (1996) 1996
Glutamate receptors are composed of subtype-specific subunits. Variation in the precise subunit composition of a receptor may result in significant functional differences. Thus, a precise knowledge of subunit composition on striatal neurons is a prerequisite for understanding the selective vulnerability of striatal neurons to excitatory amino acids. In the present study, we used an immunohistochemical double-labelling approach to localize ionotropic glutamate receptor subunits (NMDAR1, GluR1, GluR2/3, GluR4 and GluR5/6/7) on specific striatal neuron populations. Our results showed that striatal cholinergic and somatostatin interneurons were not labelled for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate, receptor subunits GluR1, GluR2/3 and GluR4. Most cholinergic and somatostatin interneurons (83.3% to 100%), however, were double-labelled for the N-methyl-D-aspartate receptor subunit NR1 and kainic acid receptor subunits GluR5/6/7. All parvalbumin interneurons were labelled for GluR1 and GluR4, and 96% GluR1 positive and 95% GluR4 positive neurons were also double-labelled as parvalbumin interneurons. About half of all parvalbumin interneurons co-localized with GluR2/3, and over 97% were labelled for NR1 and GluR5/6/7. Among striatal projection neurons, enkephalin-positive (mainly striatopallidal) neurons, striatonigral neurons (mainly substance P-positive) and calbindin-positive matrix neurons were not immunostained for GluR1 or GluR4. In contrast, 95% to 100% of each of these types of projection neurons were double-labelled for NR1, GluR2/3 and GluR5/6/7. Our results demonstrate that striatal neuron types differ in their expression of ionotropic glutamate receptor subunits and subtypes. The clear difference between striatal interneurons and projection neurons in ionotropic glutamate receptor subtypes/subunits supports the idea that differential glutamate receptor expression mechanism may account for the selective vulnerability of striatal projection neurons to excitotoxicity, and that glutamate receptor-mediated excitotoxicity may be involved in the striatal neurodegenerative diseases.
|Glutamate receptor subtypes localize to patches in the developing striatum. |
Snyder-Keller, A and Costantini, L C
Brain Res. Dev. Brain Res., 94: 246-50 (1996) 1996
The distribution of glutamate receptors in the developing striatum of the rat was studied using antibodies specific to AMPA and NMDA subtypes. Immunocytochemistry revealed a greater density of GluR1, GluR2/3, NMDAR1, and NMDAR2A/2B receptors in patches that matched the patches of substance P-immunoreactive neurons and dopaminergic terminals. GluR1-immunoreactive patches were the most distinctive and were present already at embryonic day 19.
|AMPA receptors in the rat and primate hippocampus: a possible absence of GluR2/3 subunits in most interneurons. |
Leranth, C, et al.
Neuroscience, 70: 631-52 (1996) 1996
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.
|Localization and activation of glutamate receptors in unmyelinated axons of rat glabrous skin. |
Carlton, S M, et al.
Neurosci. Lett., 197: 25-8 (1995) 1995
Immunohistochemical staining for the glutamate receptor subtypes N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) results in a significant number of labeled unmyelinated axons in the glabrous skin of the rat hindpaw. Injection of glutamate into the rat hindpaw results in behavioral changes interpreted as mechanical allodynia and mechanical hyperalgesia. The anatomical findings provide a reasonable explanation for the action of the exogenous peripheral glutamate, namely that activation of these receptors leads to increased primary afferent activity in unmyelinated axons and thus to pain behaviors. AMPA receptors are frequently associated with small clear vesicles in the axoplasm of the unmyelinated axons, many of which have been previously shown to contain high concentrations of glutamate. This finding indicates that these might be autoreceptors and so glutamate itself might regulate certain types of peripheral impulse traffic. The presence of peripheral glutamate receptors associated with unmyelinated axons suggests the possibility that glutamate antagonists applied peripherally might prevent or attenuate some pain-related behaviors.
|Differential localization of NMDA and AMPA receptor subunits in the lateral and basal nuclei of the amygdala: a light and electron microscopic study. |
Farb, C R, et al.
J. Comp. Neurol., 362: 86-108 (1995) 1995
Anatomical and physiological studies indicate that the amino acid L-glutamate is the excitatory transmitter in sensory afferent pathways to the amygdala and in intraamygdala circuits involving the lateral and basal nuclei. The regional, cellular, and subcellular immunocytochemical localizations of N-methyl-D-aspartate (NMDA) and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), two major classes of glutamate receptors, were examined in these areas of the amygdala. A monoclonal antibody and a polyclonal antiserum directed against the R1 subunit of the NMDA receptor were used. Each immunoreagent produced distinct distributions of perikaryal and neuropilar staining. Dendritic immunoreactivity was localized primarily to asymmetric (excitatory) synaptic junctions, mostly on spines, consistent with the conventional view of the organization and function of NMDA receptors. Whereas the anti-NMDAR1 antiserum produced sparse presynaptic axon terminal labeling and extensive glial labeling, the anti-NMDAR1 antibody labeled considerably fewer glia and many more presynaptic axon terminals. Labeled presynaptic terminals formed asymmetric and symmetric synapses, suggesting presynaptic regulation of both excitatory and inhibitory transmission. Immunoreactivity for different subunits of the AMPA receptor (GluR1, GluR2/3, and GluR4) was uniquely distributed across neuronal populations, and some receptor subunits were specific to certain cell types. Immunoreactivity for GluR1 and Glu2/3 was predominantely localized to dendritic shafts and was more extensive than that of GluR4 due to heavy labeling of proximal portions of dendrites. The distribution of GluR4 immunoreactivity was similar to NMDAR1: GluR4 was seen in presynaptic terminals, glia, and dendrites and was primarily localized to spines. The presynaptic localization of GluR4 in the absence of GluR2 suggests glutamate-mediated modulation of presynaptic Ca++ concentrations. These data add to our understanding of the morphological basis of pre- and postsynaptic transmission mechanisms and synaptic plasticity in the amygdala.
|Selective RNA editing and subunit assembly of native glutamate receptors. |
Puchalski, R B, et al.
Neuron, 13: 131-47 (1994) 1994
RNA editing and subunit assembly of ionotropic glutamate receptors (GluRs) were examined in an oligodendrocyte progenitor cell line, CG4, which expresses GluR2-GluR4, GluR6, GluR7, KA1, and KA2. AMPA-evoked currents rapidly desensitize, whereas kainate-evoked currents contain a steady-state component with a nearly linear current-voltage relation and a fast desensitizing component that is inwardly rectifying. The Q/R site is edited > 95% to the arginine codon in GluR2(Q607) mRNA, and < 5% in GluR6(Q621) mRNA. Immunoprecipitation experiments demonstrate that GluR6 and/or GluR7 subunits assemble with KA2, but not with GluR2-GluR4. These results indicate that oligodendrocyte progenitor cells selectively edit and assemble glutamate receptors into at least two functionally and structurally distinct heteromeric channels.
|Molecular neurobiology of glutamate receptors. |
Gasic, G P and Hollmann, M
Annu. Rev. Physiol., 54: 507-36 (1992) 1992
|Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain. |
Petralia, R S and Wenthold, R J
J. Comp. Neurol., 318: 329-54 (1992) 1992
Since four AMPA-type excitatory amino acid receptor subunits have been cloned recently, it is now possible to localize these important molecules in the nervous system. A comprehensive study of AMPA receptor immunocytochemistry was carried out on vibratome sections of rat brain, which were immunolabeled with antibodies made against peptides corresponding to the C-terminal portions of AMPA-receptor subunits: GluR1, GluR2/3, and GluR4. Labeling was most prominent in forebrain structures such as the olfactory bulb and tubercle, septal nuclei, amygdaloid complex, hippocampus, induseum griseum, habenula, and interpeduncular nucleus, and in the cerebellum. Different patterns of immunolabeling were evident with the antibodies to the four subunits, with marked contrast between densely and lightly stained structures with antibody to GluR1, widespread dense staining with antibody to GluR2/3, and moderate staining with antibody to GluR4. In the parietal cortex, some non-pyramidal neurons were more densely stained than pyramidal cells with antibodies to GluR1. Neurons of the main olfactory bulb, other than granule cells, were most densely stained with antibody to GluR1. In the cerebellum, Bergmann glia were densely stained with antibodies to GluR1 and 4, while neurons, other than granule cells, were most densely stained with antibody to GluR2/3. Immunolabeling patterns of all antibodies were consistent with that of previous in situ hybridization histochemistry studies and with the overall pattern of 3H-AMPA binding. Electron microscopy of thin sections taken from immunolabeled vibratome sections of hippocampus and cerebral cortex showed staining which was restricted mainly to postsynaptic densities and adjacent dendritoplasm, and to neuron cell body cytoplasm. We saw no convincing examples of stained presynaptic terminals, and only limited evidence of glial staining, excepting Bergmann glia.
|Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain. |
Wenthold, R J, et al.
J. Biol. Chem., 267: 501-7 (1992) 1992
Antibodies were made to synthetic peptides corresponding to sequences specific to the glutamate receptor (GluR) subunits, GluR1-4. The specificity of the antibodies was established by Western blotting using membranes of simian kidney cells (COS-7) transfected with GluR subunit DNA. Four antibodies were found to be selective for each of the four GluR subunits, and a fifth antibody recognized both GluR2 and 3. All five antibodies immunoadsorbed Triton X-100-solubilized rat brain [3H]AMPA binding activity and labeled an Mr = 108,000 band in samples of rat brain. The structure of the Triton X-100-solubilized GluR was studied using subunit-specific antibodies covalently attached to protein A-agarose and analyzing GluR subunits bound to the antibodies by Western blotting. Each of the four subunit-specific antibodies immunoadsorbed its respective GluR subunit as well as the other three forms of GluR, showing that the detergent solubilized GluR exists as hetero-oligomers composed of two or more of the four subunits. Evidence supporting a similar structure for membrane bound GluR was obtained using synaptic membranes chemically cross-linked with dithiobis(succinimidylpropionate). GluR was immunoaffinity-purified using the GluR2 and 3-selective antibody. This antibody, covalently attached to protein A-agarose, adsorbed 55% of [3H]AMPA binding activity, and after elution with 1 M KSCN, 22-37% of the binding activity was recovered. Analysis of the purified product showed a major immunoreactive band at Mr = 108,000, and silver staining identified the same major band and no additional polypeptides. The GluR receptor complex, therefore, appears to be made up exclusively of GluR1-4. In the purified GluR preparation, in addition to the Mr = 108,000 band, three higher molecular weight immunoreactive components were also detected. These bands migrated at Mr = 325,000, 470,000, and 590,000. Similar sized proteins were seen in the cross-linked synaptic membrane sample, with the Mr = 590,000 component being substantially enriched after cross-linking. The Mr = 590,000 band is the largest component detected, and it has a size consistent with its being a pentamer of the Mr = 108,000 protein.