Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Av, Gp, H, M, R, Rb||IH(P), ICC, IHC, IP, WB||Rb||Affinity Purified||Polyclonal Antibody|
|Description||Anti-NMDAR2A&B Antibody, pan|
|Presentation||Affinity purified using the immunogen peptide. Lyophilized from PBS containing 1% BSA.
Note: lot 0509010940 was shipped in liquid format; Do NOT add additional water to this lot of material.
|Safety Information according to GHS|
|Material Size||50 µg|
References | 36 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|High prevalence of NMDA receptor IgA/IgM antibodies in different dementia types. |
Doss, S; Wandinger, KP; Hyman, BT; Panzer, JA; Synofzik, M; Dickerson, B; Mollenhauer, B; Scherzer, CR; Ivinson, AJ; Finke, C; Schöls, L; Müller Vom Hagen, J; Trenkwalder, C; Jahn, H; Höltje, M; Biswal, BB; Harms, L; Ruprecht, K; Buchert, R; Höglinger, GU; Oertel, WH; Unger, MM; Körtvélyessy, P; Bittner, D; Priller, J; Spruth, EJ; Paul, F; Meisel, A; Lynch, DR; Dirnagl, U; Endres, M; Teegen, B; Probst, C; Komorowski, L; Stöcker, W; Dalmau, J; Prüss, H
Annals of clinical and translational neurology 1 822-32 2014
To retrospectively determine the frequency of N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) autoantibodies in patients with different forms of dementia.Clinical characterization of 660 patients with dementia, neurodegenerative disease without dementia, other neurological disorders and age-matched healthy controls combined with retrospective analysis of serum or cerebrospinal fluid (CSF) for the presence of NMDAR antibodies. Antibody binding to receptor mutants and the effect of immunotherapy were determined in a subgroup of patients.Serum NMDAR antibodies of IgM, IgA, or IgG subtypes were detected in 16.1% of 286 dementia patients (9.5% IgM, 4.9% IgA, and 1.7% IgG) and in 2.8% of 217 cognitively healthy controls (1.9% IgM and 0.9% IgA). Antibodies were rarely found in CSF. The highest prevalence of serum antibodies was detected in patients with "unclassified dementia" followed by progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease-related dementia, and primary progressive aphasia. Among the unclassified dementia group, 60% of 20 patients had NMDAR antibodies, accompanied by higher frequency of CSF abnormalities, and subacute or fluctuating disease progression. Immunotherapy in selected prospective cases resulted in clinical stabilization, loss of antibodies, and improvement of functional imaging parameters. Epitope mapping showed varied determinants in patients with NMDAR IgA-associated cognitive decline.Serum IgA/IgM NMDAR antibodies occur in a significant number of patients with dementia. Whether these antibodies result from or contribute to the neurodegenerative disorder remains unknown, but our findings reveal a subgroup of patients with high antibody levels who can potentially benefit from immunotherapy.
|Developmentally dynamic colocalization patterns of DSCAM with adhesion and synaptic proteins in the mouse retina. |
de Andrade, GB; Kunzelman, L; Merrill, MM; Fuerst, PG
Molecular vision 20 1422-33 2014
The Down syndrome cell adhesion molecule (Dscam) gene is required for normal dendrite arborization and lamination in the mouse retina. In this study, we characterized the developmental localization of the DSCAM protein to better understand the postnatal stages of retinal development during which laminar disorganization occur in the absence of the protein.Immunohistochemistry and colocalization analysis software were used to assay the localization of the DSCAM protein during development of the retina.We found that DSCAM was initially localized diffusely throughout mouse retinal neurites but then adopted a punctate distribution. DSCAM colocalized with catenins in the adult retina but was not detected at the active zone of chemical synapses, electrical synapses, and tight junctions. Further analysis identified a wave of colocalization between DSCAM and numerous synaptic and junction proteins coinciding with synaptogenesis between bipolar and retinal ganglion cells.Research presented in this study expands our understanding of DSCAM function by characterizing its location during the development of the retina and identifies temporally regulated localization patterns as an important consideration in understanding the function of adhesion molecules in neural development.
|Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors. |
Semerdjieva, S; Abdul-Razak, HH; Salim, SS; Yáñez-Muñoz, RJ; Chen, PE; Tarabykin, V; Alifragis, P
Molecular and cellular biology 33 1442-55 2013
Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.
|GluN2B in corticostriatal circuits governs choice learning and choice shifting. |
Brigman, JL; Daut, RA; Wright, T; Gunduz-Cinar, O; Graybeal, C; Davis, MI; Jiang, Z; Saksida, LM; Jinde, S; Pease, M; Bussey, TJ; Lovinger, DM; Nakazawa, K; Holmes, A
Nature neuroscience 16 1101-10 2013
A choice that reliably produces a preferred outcome can be automated to liberate cognitive resources for other tasks. Should an outcome become less desirable, behavior must adapt in parallel or it becomes perseverative. Corticostriatal systems are known to mediate choice learning and flexibility, but the molecular mechanisms of these processes are not well understood. We integrated mouse behavioral, immunocytochemical, in vivo electrophysiological, genetic and pharmacological approaches to study choice. We found that the dorsal striatum (DS) was increasingly activated with choice learning, whereas reversal of learned choice engaged prefrontal regions. In vivo, DS neurons showed activity associated with reward anticipation and receipt that emerged with learning and relearning. Corticostriatal or striatal deletion of Grin2b (encoding the NMDA-type glutamate receptor subunit GluN2B) or DS-restricted GluN2B antagonism impaired choice learning, whereas cortical Grin2b deletion or OFC GluN2B antagonism impaired shifting. Our convergent data demonstrate how corticostriatal GluN2B circuits govern the ability to learn and shift choice behavior.
|Anti-NMDA receptor encephalitis antibody binding is dependent on amino acid identity of a small region within the GluN1 amino terminal domain. |
Gleichman, AJ; Spruce, LA; Dalmau, J; Seeholzer, SH; Lynch, DR
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 11082-94 2012
Anti-NMDA receptor (NMDAR) encephalitis is a newly identified autoimmune disorder that targets NMDARs, causing severe neurological symptoms including hallucinations, psychosis, and seizures, and may result in death (Dalmau et al., 2008). However, the exact epitope to which these antibodies bind is unknown. A clearly defined antigenic region could provide more precise testing, allow for comparison of immunogenicity between patients to explore potential clinically relevant variations, elucidate the functional effects of antibodies, and make patients' antibodies a more effective tool with which to study NMDAR function. Here, we use human CSF to explore the antigenic region of the NMDAR. We created a series of mutants within the amino terminal domain of GluN1 that change patient antibody binding in transfected cells in stereotyped ways. These mutants demonstrate that the N368/G369 region of GluN1 is crucial for the creation of immunoreactivity. Mass spectrometry experiments show that N368 is glycosylated in transfected cells and rat brain regions; however, this glycosylation is not directly required for epitope formation. Mutations of residues N368/G369 change the closed time of the receptor in single channel recordings; more frequent channel openings correlates with the degree of antibody staining, and acute antibody exposure prolongs open time of the receptor. The staining pattern of mutant receptors is similar across subgroups of patients, indicating consistent immunogenicity, although we have identified one region that has a variable role in epitope formation. These findings provide tools for detailed comparison of antibodies across patients and suggest an interaction between antibody binding and channel function.
|Hippocampal adult neurogenesis is maintained by neil3-dependent repair of oxidative DNA lesions in neural progenitor cells. |
Christine Elisabeth Regnell,Gunn Annette Hildrestrand,Yngve Sejersted,Tirill Medin,Olve Moldestad,Veslem Rolseth,Silje Zandstra Krokeide,Rajikala Suganthan,Luisa Luna,Magnar Bj,Linda H Bergersen
Cell reports 2 2012
Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single-stranded DNA of neural stem/progenitor cells, promoting neurogenesis. Adult neurogenesis is crucial for maintenance of hippocampus-dependent functions involved in behavior. Herein, behavioral studies reveal learning and memory deficits and reduced anxiety-like behavior in Neil3(-/-) mice. Neural stem/progenitor cells from aged Neil3(-/-) mice show impaired proliferative capacity and reduced DNA repair activity. Furthermore, hippocampal neurons in Neil3(-/-) mice display
|Aluminum alters NMDA receptor 1A and 2A/B expression on neonatal hippocampal neurons in rats. |
Yuan, CY; Hsu, GS; Lee, YJ
Journal of biomedical science 18 81 2011
High aluminum (Al) content in certain infant formula raises the concern of possible Al toxicity on brain development of neonates during their vulnerable period of growing. Results of in vivo study showed that Al content of brain tissues reached to 74 μM when oral intake up to 1110 μM, 10 times of that in the hi-Al infant formula.Utilizing a cultured neuron cells in vitro model, we have assessed Al influence on neuronal specific gene expression alteration by immunoblot and immunohistochemistry and neural proliferation rate changes by MTT assay.Microscopic images showed that the neurite outgrowth of hippocampal neurons increased along with the Al dosages (37, 74 μM Al (AlCl3)). MTT results also indicated that Al increased neural cell viability. On the other hand, the immunocytochemistry staining suggested that the protein expressions of NMDAR 1A and NMDAR 2A/B decreased with the Al dosages (p less than 0.05).Treated hippocampal neurons with 37 and 74 μM of Al for 14 days increased neural cell viability, but hampered NMDAR 1A and NMDAR 2A/B expressions. It was suggested that Al exposure might alter the development of hippocampal neurons in neonatal rats.
|Neuroprotective effects of quercetin, rutin and okra (Abelmoschus esculentus Linn.) in dexamethasone-treated Mice. |
Tongjaroenbuangam W, Ruksee N, Chantiratikul P, Pakdeenarong N, Kongbuntad W, Govitrapong P
Neurochemistry international 2011
The administration of dexamethasone, a synthetic glucocorticoid receptor agonist, causes neuronal death in the CA3 layer of the hippocampus, which has been associated with learning and memory impairments. This study aimed to examine the ability of okra (Abelmoschus esculentus Linn.) extract and its derivatives (quercetin and rutin) to protect neuronal function and improve learning and memory deficits in mice subjected to dexamethasone treatment. Learning and memory functions in mice were examined using the Morris water maze test. The results showed that the mice treated with dexamethasone had prolonged water maze performance latencies and shorter time spent in the target quadrant while mice pretreated with quercetin, rutin or okra extract prior to dexamethasone treatment showed shorter latencies and longer time spent in target quadrant. Morphological changes in pyramidal neurons were observed in the dexamethasone treated group. The number of CA3 hippocampal neurons was significantly lower while pretreated with quercetin, rutin or okra attenuated this change. Prolonged treatment with dexamethasone altered NMDA receptor expression in the hippocampus. Pretreatment with quercetin, rutin or okra extract prevented the reduction in NMDA receptor expression. Dentate gyrus (DG) cell proliferation was examined using the 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry technique. The number of BrdU-immunopositive cells was significantly reduced in dexamethasone-treated mice compared to control mice. Pretreatment with okra extract, either quercetin or rutin was found to restore BrdU-immunoreactivity in the dentate gyrus. These findings suggest that quercetin, rutin and okra extract treatments reversed cognitive deficits, including impaired dentate gyrus (DG) cell proliferation, and protected against morphological changes in the CA3 region in dexamethasone-treated mice. The precise mechanism of the neuroprotective effect of these plant extracts should be further investigated.Copyright © 2011 Elsevier B.V. All rights reserved.
|Glutamate co-transmission from developing medial nucleus of the trapezoid body--lateral superior olive synapses is cochlear dependent in kanamycin-treated rats. |
Lee JH, Pradhan J, Maskey D, Park KS, Hong SH, Suh MW, Kim MJ, Ahn SC
Biochem Biophys Res Commun 405 162-7. Epub 2011 Jan 5. 2011
Cochlear dependency of glutamate co-transmission at the medial nucleus of the trapezoid body (MNTB)--the lateral superior olive (LSO) synapses was investigated using developing rats treated with high dose kanamycin. Rats were treated with kanamycin from postnatal day (P) 3 to P8. A scanning electron microscopic study on P9 demonstrated partial cochlear hair cell damage. A whole cell voltage clamp experiment demonstrated the increased glutamatergic portion of postsynaptic currents (PSCs) elicited by MNTB stimulation in P9-P11 kanamycin-treated rats. The enhanced VGLUT3 immunoreactivities (IRs) in kanamycin-treated rats and asymmetric VGLUT3 IRs in the LSO of unilaterally cochlear ablated rats supported the electrophysiologic data. Taken together, it is concluded that glutamate co-transmission is cochlear-dependent and enhanced glutamate co-transmission in kanamycin-treated rats is induced by partial cochlear damage.Copyright © 2011 Elsevier Inc. All rights reserved.
|Visual experience-independent functional expression of NMDA receptors in the developing rabbit retina. |
Chang, YC; Chen, CY; Chiao, CC
Investigative ophthalmology & visual science 51 2744-54 2010
Activation of the NMDA glutamate receptors is critical for the initiation of synaptic plasticity. In the developing rat retina, NMDA receptor function has been associated with visual experience, though the light-dependent regulation of the subunit composition of the NMDA receptors is controversial. In the present study, the functional expression of NMDA receptors in the developing rabbit retina was characterized and the impact of light deprivation on how the subunit composition of NMDA receptors is regulated was examined.Antibodies against NR1 and NR2A/B were used to examine neonatal expression patterns of the NMDA receptor subunits. Furthermore, the functional NMDA receptors were mapped using the agmatine (AGB) activation assay.Although NR1 and NR2A/B subunit immunoreactivity was prominently detectable only immediately after birth, AGB activation assay showed that functional NMDA receptors could be identified as early as embryonic day 21. No significant difference was observed between normal- and dark-reared animals in terms of their NR1 and NR2A/B expression. In addition, a comparison of AGB permeation between normal- and dark-reared animals showed no difference in functional expression of NMDA receptors.These results indicate that NMDA receptors participate in the synaptic maturation of retinal circuits during the early stages of development but that the functional NMDA receptors, including their subunit composition, in the developing rabbit retina are independent of the rabbit's visual experience.
|Endoplasmic reticulum-associated degradation of the NR1 but not the NR2 subunits of the N-methyl-D-aspartate receptor induced by inhibition of the N-glycosylation in cortical neurons |
Gascón, Sergio, et al.
J. Neurosci. Res., 85:1713-1723 (2007) 2007
|NMDA inhibitors cause apoptosis of pyramidal neurons in mature piriform cortex: evidence for a nitric oxide-mediated effect involving inhibitory interneurons. |
Lijun Zhou,Annie M Welsh,David Chen,Vassilis E Koliatsos
Neuropharmacology 52 2007
Pyramidal relay neurons in limbic cortex are vulnerable to denervation lesions, i.e. pyramidal neurons in layer IIalpha of piriform cortex undergo transsynaptic apoptosis after lesions that interrupt their inputs from the olfactory bulb. We have previously established the role of inhibitory interneurons in elaborating signals that lead to the apoptosis of projection neurons in these lesion models, i.e. the upregulation of neuronal NOS and release of nitric oxide. Thus, we have proposed that cortical interneurons play an essential role in transducing injury to degenerative effects for nearby pyramidal neurons. In the present study, we extend the previous findings to a toxic model of degeneration of pyramidal neurons in the adult paralimbic cortex, i.e. after exposure to the NMDA channel blocker MK801. Our findings indicate that treatment of adult rats with MK801 in doses previously found to cause alterations in pyramidal neurons of the retrosplenial cortex (5mg/kg) results in an active caspase 3 (+), ultrastructurally apoptotic type of cell death involving the same projection neurons of layer IIalpha that are also vulnerable to bulbotomy lesions. Interneurons of layer I are induced by MK801 treatment to higher levels of nNOS expression and the selective nNOS inhibitor BRNI ameliorates pyramidal cell apoptosis caused by MK801. Our results indicate that certain pyramidal neurons in piriform cortex are very sensitive to NMDA blockade as they are to disconnection from modality-specific afferents and that inhibitory interneurons play significant roles in mediating various types of pro-apoptotic insults to cortical projection neurons via nNOS/NO signaling.Full Text Article
|NMDA receptor contribution to the climbing fiber response in the adult mouse Purkinje cell. |
Piochon, C; Irinopoulou, T; Brusciano, D; Bailly, Y; Mariani, J; Levenes, C
The Journal of neuroscience : the official journal of the Society for Neuroscience 27 10797-809 2007
Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at approximately 21 d after birth and control the complex spike waveform.
|Integrins control dendritic spine plasticity in hippocampal neurons through NMDA receptor and Ca2+/calmodulin-dependent protein kinase II-mediated actin reorganization. |
Shi, Y; Ethell, IM
The Journal of neuroscience : the official journal of the Society for Neuroscience 26 1813-22 2006
The formation of dendritic spines during development and their structural plasticity in the adult brain are critical aspects of synaptogenesis and synaptic plasticity. Many different factors and proteins have been shown to control dendritic spine development and remodeling (Ethell and Pasquale, 2005). The extracellular matrix (ECM) components and their cell surface receptors, integrins, have been found in the vicinity of synapses and shown to regulate synaptic efficacy and play an important role in long-term potentiation (Bahr et al., 1997; Chavis and Westbrook, 2001; Chan et al., 2003; Lin et al., 2003; Bernard-Trifilo et al., 2005). Although molecular mechanisms by which integrins affect synaptic efficacy have begun to emerge, their role in structural plasticity is poorly understood. Here, we show that integrins are involved in spine remodeling in cultured hippocampal neurons. The treatment of 14 d in vitro hippocampal neurons with arginine-glycine-aspartate (RGD)-containing peptide, an established integrin ligand, induced elongation of existing dendritic spines and promoted formation of new filopodia. These effects were also accompanied by integrin-dependent actin reorganization and synapse remodeling, which were partially inhibited by function-blocking antibodies against beta1 and beta3 integrins. This actin reorganization was blocked with the NMDA receptor (NMDAR) antagonist MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate]. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide) also suppressed RGD-induced actin reorganization and synapse remodeling. Our findings show that integrins control ECM-mediated spine remodeling in hippocampal neurons through NMDAR/CaMKII-dependent actin reorganization.
|Novel subcellular distribution pattern of A-type K+ channels on neuronal surface. |
Kollo, M; Holderith, NB; Nusser, Z
The Journal of neuroscience : the official journal of the Society for Neuroscience 26 2684-91 2006
Potassium channels comprise the most diverse family of ion channels. In nerve cells, their critical roles in synaptic integration and output generation have been demonstrated. Here, we provide evidence for a distribution that predicts a novel role of K+ channels in the CNS. Our experiments revealed a highly selective clustering of the Kv4.3 A-type K+ channel subunits at specialized junctions between climbing fibers and cerebellar GABAergic interneurons. High-resolution ultrastructural and immunohistochemical experiments demonstrated that these junctions are distinct from known chemical and electrical (gap junctions) synapses and also from puncta adherentia. Each cerebellar interneuron contains many such K+ channel-rich specializations, which seem to be distributed throughout the somatodendritic surface. We also show that such K+ channel-rich specializations are not only present in the cerebellum but are widespread in the rat CNS. For example, mitral cells of the main olfactory bulb establish Kv4.2 subunit-positive specializations with each other. At these specializations, both apposing membranes have a high density of K+ channels, indicating bidirectional signaling. Similar specializations with pronounced coclustering of the Kv4.2 and 4.3 subunits were observed between nerve cells in the medial nucleus of the habenula. Based on our results and on the known properties of A-type K+ channels, we propose that strategically clustered K+ channels at unique membrane specializations could mediate a novel type of communication between nerve cells.Full Text Article
|Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology. |
Joie A Bernard-Trifilo, Enikö A Kramár, Reidun Torp, Ching-Yi Lin, Eduardo A Pineda, Gary Lynch, Christine M Gall
Journal of neurochemistry 93 834-49 2005
Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline-rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to beta1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin-mediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and beta1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.
|Regulation of N-methyl-D-aspartate receptor subunit expression in the fetal guinea pig brain. |
Owen, D; Setiawan, E; Li, A; McCabe, L; Matthews, SG
Biology of reproduction 71 676-83 2004
N-methyl-d-aspartate receptors (NMDARs) are critical for neuronal maturation and synaptic formation as well as for the onset of long-term potentiation, a process critical to learning and memory in postnatal life. In the current study, we demonstrated that NMDAR subunits undergo spatial, temporal, and sex-specific regulation. During development, we observed increasing NR1 and NR2A expression at the same time as levels of NR2B subunits decreased in the hippocampus and cortex in the fetal guinea pig. We have also shown that glucocorticoids can modulate fetal NMDAR subunit expression in a sex-specific fashion. This is clinically important because synthetic glucocorticoids are administered to pregnant women at risk of preterm labor. Repeated exposure to exogenous glucocorticoids caused a dose-dependent decrease in NR1 mRNA levels and increased NR2A mRNA expression in the female hippocampus at Gestational Day 62. There are significant changes in NMDAR subunit expression in late gestation. It is possible that these alter NMDA-dependent signaling at this time. Prenatal exposure to exogenous glucocorticoids modifies the trajectory of NMDAR subunit expression in females but not in males.
|Western Blotting||Guinea Pig||15115726|
|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.
|Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus. |
Henkemeyer, M; Itkis, OS; Ngo, M; Hickmott, PW; Ethell, IM
The Journal of cell biology 163 1313-26 2003
Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2-mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.
|Proteolysis of the N-methyl-d-aspartate receptor by calpain in situ. |
Guttmann, RP; Sokol, S; Baker, DL; Simpkins, KL; Dong, Y; Lynch, DR
The Journal of pharmacology and experimental therapeutics 302 1023-30 2002
N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of (125)I-MK-801 ((125)I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.
|Assembly with the NR1 subunit is required for surface expression of NR3A-containing NMDA receptors. |
Perez-Otano, I, et al.
J. Neurosci., 21: 1228-37 (2001) 2001
Functional NMDA receptors are heteromultimeric complexes of the NR1 subunit in combination with at least one of the four NR2 subunits (A-D). Coexpression of NR3A, an additional subunit of the NMDA receptor family, modifies NMDA-mediated responses. It is unclear whether NR3A interacts directly with NR1 and/or NR2 subunits and how such association might regulate the intracellular trafficking and membrane expression of NR3A. Here we show that NR3A coassembles with NR1-1a and NR2A to form a receptor complex with distinct single-channel properties and a reduced relative calcium permeability. NR3A associates independently with both NR1-1a and NR2A in the endoplasmic reticulum, but only heteromeric complexes containing the NR1-1a NMDA receptor subunit are targeted to the plasma membrane. Homomeric NR3A complexes or complexes composed of NR2A and NR3A were not detected on the cell surface and are retained in the endoplasmic reticulum. Coexpression of NR1-1a facilitates the surface expression of NR3A-containing receptors, reduces the accumulation of NR3A subunits in the endoplasmic reticulum, and induces the appearance of intracellular clusters where both subunits are colocalized. Our data demonstrate a role for subunit oligomerization and specifically assembly with the NR1 subunit in the trafficking and plasma membrane targeting of the receptor complex.
|The NR1 subunit of the N-methyl-D-aspartate receptor can be efficiently expressed alone in the cell surface of mammalian cells and is required for the transport of the NR2A subunit. |
García-Gallo, M, et al.
Biochem. J., 356: 539-47 (2001) 2001
We have used a heterologous system of expression of N-methyl-D-aspartate (NMDA) receptors based on the use of vaccinia virus to analyse the maturation, transport, assembly and differential expression of the NR1 and NR2A subunits of the receptors. We have demonstrated that the NR1 subunit is efficiently transported to the plasma membrane in cells expressing NR1 alone, similarly to cells producing NR1 and NR2A together. In contrast, NR2A requires NR1 expression to be located at the cell surface. The stability of both receptor subunits expressed alone is similar to that obtained in cells producing NR1 and NR2A. In pulse-chase experiments, the NR1 subunit displays a biphasic decay, with a fraction of the protein having a half-life of only 1 h and the remaining presenting a turnover longer than 24 h, similar to values obtained for the NR2A subunit. Our results also show a maturation process affecting the carbohydrate moiety in the NR1 subunit, such that immature NR1 has a much shorter half-life than the mature form or the NR2A subunit. Finally, we show that only a fraction of mature NR1 interacts with NR2A to form multimeric functional complexes.
|Laminar organization of the NMDA receptor complex within the postsynaptic density. |
J G Valtschanoff, R J Weinberg, J G Valtschanoff, R J Weinberg
The Journal of neuroscience : the official journal of the Society for Neuroscience 21 1211-7 2001
The NR2 subunit is an essential component of the NMDA receptor. Recent biochemical research has identified a number of molecules that can bind directly or indirectly to its cytoplasmic tail. These postsynaptic density (PSD) proteins play a role in intracellular signal transduction, and are implicated in synaptic plasticity and memory mechanisms. We performed systematic electron microscopic immunogold analysis in rat neocortex to determine the spatial organization of NR2, in relation to six other proteins thought to be involved in the NMDA receptor complex. Peak concentrations of each protein were within the PSD but in different layers of the density. In the axodendritic axis, gold particles coding for PSD-95 lay an average of 12 nm cytoplasmic to the extracellular face of the plasma membrane, very close to the C terminal of NR2. Nitric oxide synthase lay 18 nm inside the membrane; the scaffolding proteins guanylate kinase-associated protein and Shank lay 24-26 nm inside the membrane; and CRIPT and dynein light chain, proteins that may link the complex to cytoskeletal elements, lay on the cytoplasmic side of the PSD, 29-32 nm inside the plasma membrane and extending into the spine cytoplasm. The supramolecular organization of these molecules may modulate intracellular transduction of NMDA-mediated signals.
|Specific proteolysis of the NR2 subunit at multiple sites by calpain. |
R P Guttmann, D L Baker, K M Seifert, A S Cohen, D A Coulter, D R Lynch
Journal of neurochemistry 78 1083-93 2001
The NMDA subtype of glutamate receptor plays an important role in the molecular mechanisms of learning, memory and excitotoxicity. NMDA receptors are highly permeable to calcium, which can lead to the activation of the calcium-dependent protease, calpain. In the present study, the ability of calpain to modulate NMDA receptor function through direct proteolytic digestion of the individual NMDA receptor subunits was examined. HEK293t cells were cotransfected with the NR1a/2A, NR1a/2B or NR1a/2C receptor combinations. Cellular homogenates of these receptor combinations were prepared and digested by purified calpain I in vitro. All three NR2 subunits could be proteolyzed by calpain I while no actin or NR1a cleavage was observed. Based on immunoblot analysis, calpain cleavage of NR2A, NR2B and NR2C subunits was limited to their C-terminal region. In vitro calpain digestion of fusion protein constructs containing the C-terminal region of NR2A yielded two cleavage sites at amino acids 1279 and 1330. Although it has been suggested that calpain cleavage of the NMDA receptor may act as a negative feedback mechanism, the current findings demonstrated that calpain cleavage did not alter [(125)I]MK801 binding and that receptors truncated to the identified cleavage sites had peak intracellular calcium levels, (45)Ca uptake rates and basal electrophysiological properties similar to wild type.
|Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor. |
Strack, S, et al.
J. Biol. Chem., 275: 23798-806 (2000) 2000
Calcium influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor and activation of calcium/calmodulin-dependent kinase II (CaMKII) are critical events in certain forms of synaptic plasticity. We have previously shown that autophosphorylation of CaMKII induces high-affinity binding to the NR2B subunit of the NMDA receptor (Strack, S., and Colbran, R. J. (1998) J. Biol. Chem. 273, 20689-20692). Here, we show that residues 1290-1309 in the cytosolic tail of NR2B are critical for CaMKII binding and identify by site-directed mutagenesis several key residues (Lys(1292), Leu(1298), Arg(1299), Arg(1300), Gln(1301), and Ser(1303)). Phosphorylation of NR2B at Ser(1303) by CaMKII inhibits binding and promotes slow dissociation of preformed CaMKII.NR2B complexes. Peptide competition studies imply a role for the CaMKII catalytic domain, but not the substrate-binding pocket, in the association with NR2B. However, analysis of monomeric CaMKII mutants indicates that the holoenzyme structure may also be important for stable association with NR2B. Residues 1260-1316 of NR2B are sufficient to direct the subcellular localization of CaMKII in intact cells and to confer dynamic regulation by calcium influx. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B bidirectionally modulates colocalization with CaMKII after NMDA receptor activation, suggesting a dynamic model for the translocation of CaMKII to postsynaptic targets.
|Sulcogyral variation in NMDA receptor 2A/B subunit immunoreactivity in human brain. |
S M Sisodiya, J Heffernan, P J Harrison, M V Squier, M Thom
Neuroreport 11 2601-6 2000
NMDA receptors (NR) are important in many neurological processes. Using a large series of human brain tissue, we show that the distribution of NR2A/B immunoreactivity varies according to position along a gyrus. For pyramidal neurons in laminae II and III, immunoreactivity is most marked at gyral crown and gyral lips, diminishes along sulcal wall, and is barely detectable in sulcal floor cortex. In contrast, in some cases, immunoreactivity in laminae V and VI pyramidal neurons shows the reverse pattern. Neurofilament and calretinin immunoreactivity do not show this phenomenon. The findings suggest novel functional regionalization at the sulcogyral level in normal human brain.
|Synaptic localization of ionotropic glutamate receptors in the rat substantia nigra. |
Chatha, B T, et al.
Neuroscience, 101: 1037-51 (2000) 2000
Glutamatergic neurotransmission in the substantia nigra pars compacta and pars reticulata is mediated through N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxaline propionic acid/kainate (AMPA) type receptors as well as other glutamate receptors and is critical for basal ganglia functioning. A major glutamatergic input to the substantia nigra originates in the subthalamic nucleus, and the long-lasting stimulation of the dopaminergic cells of the substantia nigra pars compacta by the subthalamic neurons has been implicated in the pathophysiology of Parkinson's disease. The objectives of the present study were to determine the subcellular and subsynaptic localization of subunits of the N-methyl-D-aspartate and AMPA receptors in the substantia nigra, and also to determine whether co-localization of N-methyl-D-aspartate and AMPA receptor subunits occur at individual synapses. To achieve this, pre-embedding and post-embedding immunocytochemistry was applied to sections of substantia nigra using antibodies that recognize the NR1 and NR2A/B subunits of the N-methyl-D-aspartate receptor, and GluR2/3 subunits of the AMPA receptor.In both regions of the substantia nigra, immunolabelling for each of the subunits was observed in numerous perikarya and proximal dendrites. At the subcellular level, silver-intensified immunogold particles localizing N-methyl-D-aspartate and AMPA receptor subunits were most commonly present within dendrites where they were associated with a variety of intracellular organelles and with the internal surface of the plasma membrane. Post-embedding immunogold labelling revealed immunoparticles labelling for NR1, NR2A/B and GluR2/3 to be enriched at asymmetric synaptic specializations, although a large proportion of asymmetric synapses were immunonegative. Double immunolabelling revealed, in addition to single-labelled synapses, the co-localization of subunits of the N-methyl-D-aspartate receptor and subunits of the AMPA receptor at individual asymmetric synapses. Similarly, double immunolabelling also revealed the co-localization of the NRl and NR2A/B subunits of the N-methyl-D-aspartate receptor at individual asymmetric synapses. Labelling for NR1 and GluR2/3 was, on average, relatively evenly distributed across the width of the synapse with a gradual reduction towards the periphery when analysed in single sections.In summary, the present results demonstrate that AMPA and N-methyl-D-aspartate receptors are selectively localized at a subpopulation of asymmetric synapses in the substantia nigra pars compacta and reticulata and that the two receptor types, at least partially co-localize at individual synapses. It is concluded that glutamatergic transmission in the substantia nigra pars compacta and pars reticulata occurs primarily at asymmetric synapses and, at least in part, is mediated by both N-methyl-D-aspartate and AMPA receptors.
|Selective coexpression of NMDAR2A/B and NMDAR1 subunit proteins in dysplastic neurons of human epileptic cortex. |
Ying, Z, et al.
Exp. Neurol., 159: 409-18 (1999) 1999
NR1 and NR2 are the two gene families for the NMDA receptor. In vitro studies show that while NR2 alone is nonfunctional, NR1 alone produces weak currents to glutamate or NMDA. We previously showed by immunocytochemistry (ICC) that in normal appearing, nonepileptic human cortical neurons, only NR1 and not NR2 proteins were expressed, in contrast to the presence of both NR1 and NR2 in normal rat cortical neurons. We also showed, in dysplastic epileptic cortex, that both NR1 and NR2 were highly expressed using ICC on adjacent 30-microm sections. However, the relative coexpressions of NR1 and NR2 proteins in single neurons in single sections of human epileptic cortex were unknown. In this study, we used double-labeled immunofluorescence and confocal microscopy to examine the distribution and coexpression of subunit proteins for NR1 and NR2A/B in both nondysplastic (control comparison) and dysplastic regions of human brain resected for the treatment of intractable epilepsy (11 patients). In nondysplastic regions, cortical neurons did not have immunoreactivity (ir) for NR2A/B, whereas NR1-ir was abundant. By contrast, dysplastic neurons in the regions with epileptic cortical dysplasia showed intense NR2A/B-ir in the somata and their dendritic processes. These same NR2A/B-ir dysplastic neurons were colabeled by NR1. These results demonstrate directly that dysplastic neurons express both NR2A/B and NR1 proteins, whereas nondysplastic cortical neurons express only NR1 proteins. Selective coexpression of NR2A/B and NR1 in dysplastic neurons suggests that NR2A/B may form heteromeric NR1-NR2 coassemblies and hyperexcitability in dysplastic neurons that could contribute to focal seizure onset.
|Expression of N-methyl-D-aspartate receptors using vaccinia virus causes excitotoxic death in human kidney cells. |
García-Gallo, M, et al.
J. Cell. Biochem., 72: 135-44 (1999) 1999
N-Methyl-D-Aspartate (NMDA) receptors containing NR1 and NR2A subunits have been expressed with high efficiency in Human Embryonic Kidney 293 cells with the aid of a recombinant vaccinia virus. This expression system produced functional receptors that sustained calcium influxes dependent on receptor agonists and inhibited by receptor antagonists. Immunocytochemistry of the recombinant receptors demonstrated that they were properly arranged in membrane structures. The entrance of calcium through the recombinant receptors induced delayed toxicity, demonstrated by approximately a three-fold increase in the number of dead cells obtained 12 h after the antagonist 2-amino-phosphopentanoic acid (DL-AP5) was removed from the culture. This result correlated with more than 88% inhibition in the expression of a reporter gene 24 h after antagonist removal. Calcium toxicity was completely abolished by specific antagonists of the NMDA receptor. Treatment of cell extracts with N-glycosydase showed that both receptor subunits were N-glycosylated. Tunicamycin prevented calcium toxicity; gel electrophoresis studies showed that this protection was likely due to degradation of the NR1 subunit.
|Neuronal and glial localization of NR1 and NR2A/B subunits of the NMDA receptor in the human cerebral cortex. |
F Conti, P Barbaresi, M Melone, A Ducati
Cerebral cortex (New York, N.Y. : 1991) 9 110-20 1999
N-Methyl-D-aspartate (NMDA) receptors play a critical role in many cortical functions and are implicated in several neuropsychiatric diseases. In this study, the cellular expression of the NMDAR1 (NR1) and NMDAR2A and B (NR2A and B) subunits was investigated in the human cerebral cortex by immunocytochemistry with antibodies that recognize the NR1 or the NR2A and B subunits of the NMDA receptor. In frontal (areas 10 and 46) and temporal (area 21) association cortices and the cingulofrontal transition cortex (area 32), NR1 and NR2A/B immunoreactivity (ir) were similar and were localized to numerous neurons in all cortical layers. NR1- and NR2A/B-positive neurons were mostly pyramidal cells, but some nonpyramidal neurons were also labeled. Electron-microscopic observations showed that NR1 and NR2A/B ir were similar. In all cases, labeling of dendrites and dendritic spines was intense. In addition, both NR1 and NR2A/B were consistently found in the axoplasm of some axon terminals and in distal astrocytic processes. This investigation revealed that numerous NMDA receptors are localized to dendritic spines, and that they are also localized to axon terminals and astrocytic processes. These findings suggest that the effects of cortical NMDA activation in the human cortex do not depend exclusively on the opening of NMDA channels located at postsynaptic sites, and that the localization of NMDA receptors is similar in a variety of mammalian species.
|Assembly of proteins to postsynaptic densities after transient cerebral ischemia. |
Hu, B R, et al.
J. Neurosci., 18: 625-33 (1998) 1998
Transient ischemia leads to changes in synaptic efficacy and results in selective neuronal damage during the postischemic phase, although the mechanisms are not fully understood. The protein composition and ultrastructure of postsynaptic densities (PSDs) were studied by using a rat transient ischemic model. We found that a brief ischemic episode induced a marked accumulation in PSDs of the protein assembly ATPases, N-ethylmaleimide-sensitive fusion protein, and heat-shock cognate protein-70 as well as the BDNF receptor (trkB) and protein kinases, as determined by protein microsequencing. The changes in PSD composition were accompanied by a 2.5-fold increase in the yield of PSD protein relative to controls. Biochemical modification of PSDs correlated well with an increase in PSD thickness observed in vivo by electron microscopy. We conclude that a brief ischemic episode modifies the molecular composition and ultrastructure of synapses by assembly of proteins to the postsynaptic density, which may underlie observed changes in synaptic function and selective neuronal damage.
|NMDAR2 upregulation precedes mossy fiber sprouting in kainate rat hippocampal epilepsy. |
Mikuni, N, et al.
Neurosci. Lett., 255: 25-8 (1998) 1998
Following intrahippocampal (hilar) kainic acid (KA) lesions in rats, NMDAR2A/B receptor proteins are upregulated significantly in the inner molecular layer (IML) of the dentate gyrus by post-injection day 5. By contrast, the aberrant mossy fibers which reinnervate the IML remained in the subgranular zone before sprouting and synapsing in the IML, which occurs at approximately post-KA day 17. For 40 days thereafter, this mossy fiber ingrowth progressed, while the increased NMDAR2A/B (receptors) immunoreactivity remained at the same densities. These results suggest that new NMDAR2A/B proteins in granule cell dendrites are limited to the IML, which is the eventual site for MF hyperinnervation, neosynaptogenesis, and recurrent synaptic hyperexcitability.
|D-serine as a neuromodulator: regional and developmental localizations in rat brain glia resemble NMDA receptors. |
Schell, M J, et al.
J. Neurosci., 17: 1604-15 (1997) 1997
D-Serine is localized in mammalian brain to a discrete population of glial cells near NMDA receptors, suggesting that D-serine is an endogenous agonist of the receptor-associated glycine site. To explore this possibility, we have compared the immunohistochemical localizations of D-serine, glycine, and NMDA receptors in rat brain. In the telencephalon, D-serine is concentrated in protoplasmic astrocytes, which are abundant in neuropil in close vicinity to NMDA receptor 2A/B subunits. Ultrastructural examination of the CA1 region of hippocampus reveals D-serine in the cytosolic matrix of astrocytes that ensheath neurons and blood vessels, whereas NR2A/B is concentrated in dendritic spines. By contrast, glycine immunoreactivity in telencephalon is the lowest in brain. During postnatal week 2, D-serine levels in cerebellum are comparable to those in adult cerebral cortex but fall to undetectable levels by day 26. During week 2, we observe parallel ontogeny of D-serine in Bergmann glia and NR2A/B in Purkinje cells, suggesting a role for astrocytic D-serine in NMDA receptor-mediated synaptogenesis. D-Serine in the radial processes of Bergmann glia is also well positioned to regulate NMDA receptor-dependent granule cell migration. In the inner granule layer, D-serine is found transiently in protoplasmic astrocytes surrounding glomeruli, where it could regulate development of the mossy fiber/granule cell synapse. D-Serine seems to be the endogenous ligand of glycine sites in the telencephalon and developing cerebellum, whereas glycine predominates in the adult cerebellum, olfactory bulb, and hindbrain.
|Aberrant hippocampal mossy fiber sprouting correlates with greater NMDAR2 receptor staining. |
Mathern, G W, et al.
Neuroreport, 7: 1029-35 (1996) 1996
This study determined in temporal lobe epilepsy patients and rats injected with intrahippocampal kainate (KA) whether fascia dentata molecular layer mossy fiber sprouting was associated with increases in NMDAR2 immunoreactivity (IR). Patients with hippocampal sclerosis (n = 11) were compared with those with temporal mass lesions (n = 7) and material obtained at autopsies (n = 4); and unilateral KA-injected rat hippocampi (n = 7) were compared with the contralateral saline-injected side and non-lesioned animals (n = 7; control). Hippocampi were studied for neo-Timm's stained mossy fiber sprouting and NMDAR2 IR. The staining was quantified as gray values (GV) using computer image analysis. Hippocampal sclerosis patients and KA-injected rats showed the greatest inner molecular layer (IML) mossy fiber sprouting and NMDAR2 staining. Compared with autopsies and patients with mass lesions, hippocampal sclerosis patients had greater IML neo-Timm's (p = 0.0018) and NMDAR2 staining (p = 0.0063). Similarly, compared with controls and saline-injected rats, KA-injected hippocampi showed greater IML mossy fiber sprouting and NMDAR2 IR (p = 0.0001). Furthermore, IML mossy fiber sprouting positively correlated with greater IML NMDAR2 staining in both human and experimental rat groups (p < 0.0099). These results support the hypothesis that in severely damaged hippocampi abnormal mossy fiber sprouting and concordant increases in IML NMDAR2 receptor staining may contribute or partially explain granule cell hyperexcitability and the pathophysiology of hippocampal epilepsy.
|NMDA receptor upregulation: molecular studies in cultured mouse cortical neurons after chronic antagonist exposure. |
P Follesa, M K Ticku
The Journal of neuroscience : the official journal of the Society for Neuroscience 16 2172-8 1996
We examined the possibility of changes in gene expression of the NMDA receptor subunits after chronic antagonist treatment. Exposure of neurons to the NMDA antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP-5) produced an increase in the levels of the R2B mRNA subunit. Concomitant exposure of neurons to AP-5 and NMDA reversed the upregulation. Chronic AP-5 treatment increased the R1 polypeptide, whereas no change was observed in the levels of mRNA encoding the R1 subunit. A more pronounced increase was observed in the R2A/B polypeptides. These data demonstrate that chronic treatment with NMDA antagonists selectively upregulates the NMDA receptor mRNAs and polypeptides. Furthermore, antagonist treatment produced a differential regulation of the R1, R2A, and R2B subunits in cortical neurons.
|The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1. |
Petralia, R S, et al.
J. Neurosci., 14: 6102-20 (1994) 1994
Neuronal plasticity associated with learning, memory and development is controlled, in part, by NMDA receptors, which are complexes consisting of the subunit NMDAR1 (NR1) and one or more NMDAR2 subunits (NR2A-NR2D). We made a polyclonal antibody to a C-terminus peptide of NR2A. In analysis of transfected cell membranes, this antibody recognizes NR2A and NR2B, and to a slight extent, NR2C and NR2D. In Western blots of rat brain, the antibody labeled a single band that comigrated with NR2A and NR2B. This antibody (NR2A/B) did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained sections of rat brain showed significant staining throughout the nervous system, including olfactory bulb, cerebral cortex, hippocampus, caudate-putamen, and many brainstem nuclei, as well as in neurons of spinal cord and sensory ganglia. This widespread distribution of staining was similar to that found with an antibody to NR1, supporting the presence of functional NR1/NR2 complexes throughout the nervous system. In the cerebellum, in contrast to staining with NR1 antibody, Purkinje cell staining with NR2A/B antibody was low, indicating that these neurons may lack functional NMDA receptors. EM examination revealed dense staining in dendrites and postsynaptic densities in cerebral cortex and hippocampus, similar to those seen with antibody to NR1. Since functional NMDA receptor complexes at synapses appear to require both NR1 and NR2 subunit proteins for full function, this study provides structural evidence for functional NR1/NR2 receptors in vivo in the nervous system.
|Pathways and Biomarkers of Glutamatergic Synapse Flyer (EMD)|
|Anti-NMDAR2A&B, pain - Data Sheet|
|Anti-NMDAR2A&B, pan - Data Sheet|