NR2A Expression Research Articles

NMDA receptor–mediated regulation of human megakaryocytopoiesis

AbstractIdentification of the regulatory inputs that direct megakaryocytopoiesis and platelet production is essential for the development of novel therapeutic strategies for the treatment of thrombosis and related hematologic disorders. We have previously shown that primary human megakaryocytes express theN-methyl-d-aspartate acid (NMDA) receptor 1 (NR1) subunit of NMDA-type glutamate receptors, which appear to be pharmacologically similar to those identified at neuronal synapses, responsible for mediating excitatory neurotransmission in the central nervous system. However, the functional role of NMDA receptor signaling in megakaryocytopoiesis remains unclear. Here we provide evidence that demonstrates the fundamental importance of this signaling pathway during human megakaryocyte maturation in vitro. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of RNA extracted from CD34+-derived megakaryocytes identified expression of NR2A and NR2D receptor subunits in these cells, as well as the NMDA receptor accessory proteins, Yotiao and postsynaptic density protein 95 (PSD-95). In functional studies, addition of a selective NMDA receptor antagonist, MK-801 inhibited proplatelet formation, without affecting proliferation or apoptosis. Exposure of CD34+cells to MK-801 cultured for 14 days in the presence of thrombopoietin induced a decrease in expression of the megakaryocyte cell surface markers CD61, CD41a, and CD42a compared with controls. At an ultrastructural level, MK-801–treated cells lacked α-granules, demarcated membranes, and multilobed nuclei, which were prominent in untreated mature megakaryocyte controls. Using immunohistochemistry on sections of whole tibiae from c-Mpl knockout mice we demonstrated that megakaryocytic NMDA receptor expression was maintained following c-Mpl ablation. These data support a fundamental role for glutamate signaling in megakaryocytopoiesis and platelet production, which is likely to be independent of thrombopoietin-mediated effects.

Ethanol-induced alteration in N-methyl- d-aspartate receptor 2A C-terminus and protein kinase C activity in rat brain

Ethanol is a potent inhibitor of the N-methyl- d-aspartate (NMDA) subtype of glutamate receptor. In the present study, expression of NR2A and its phosphorylation status were investigated in adult rat cerebral cortex and cerebellum, using an experimental paradigm of in vivo chronic ethanol exposure. In addition, PKC activity was measured in both cytosol and membrane fraction of cerebral cortex and cerebellum using Histone III S as substrate. Western blot analysis using NR2A antibody showed an increased immunoreactivity in cerebral cortex and no immunoreactivity in cerebellum of alcohol-treated rats. Furthermore, PKC activity was increased in both membrane and cytosolic fraction of alcohol-treated rat cerebellum, whereas PKC activity in cerebral cortex was found to be decreased in membrane fraction with no appreciable change in cytosolic fraction. In vitro phosphorylation study showed hypophosphorylation in ethanol-treated cerebral cortex and cerebellum. Our current findings imply that the truncation of NR2A subunit upon alcohol administration in cerebellum probably contributes to altered NMDA receptor function and cerebellar atrophy and motor incoordination in alcoholic rats.

Assembly of N-methyl-D-aspartate (NMDA) receptors.

The N-methyl-D-aspartate receptor (NMDAR) requires both NR1 and NR2 subunits to form a functional ion channel. Despite the recent advances in our understanding of the contributions of these different subunits to both the function and pharmacology of the NMDAR, the precise subunit stoichiometry of the receptor and the regions of the subunits governing subunit interactions remain unclear. Since NR2 subunits are not transported to the cell surface unless they associate with NR1 subunits, cell-surface expression of NR2A can be used to monitor the association of the different subunits in cells transfected with N- and C-terminally truncated NR1 subunits. By combining measurements of cell-surface expression of NR2A with co-immunoprecipitation experiments, and by using Blue Native gel electrophoresis to determine the oligomerization status of the subunits, we have shown that regions of the N-terminus of NR1 are critical for subunit association, whereas the truncation of the C-terminus of NR1 before the last transmembrane region has no effect on the association of the subunits. Evidence from the Blue Native gels, sucrose-gradient centrifugation and size exclusion of soluble NR1 domains suggests that NR1 subunits alone can form stable dimers. Using a cell line, which can be induced to express the NMDAR following exposure to dexamethasone, we have shown that NMDARs can be expressed at the cell surface within 5 h of the recombinant gene induction, and that there appears to be a delay between the first appearance of the subunits and their stable association.

Impaired NMDA Receptor-Mediated Postsynaptic Function and Blunted NMDA Receptor-Dependent Persistent Pain in Mice Lacking Postsynaptic Density-93 Protein

Modification of synaptic NMDA receptor (NMDAR) expression influences NMDAR-mediated synaptic function and associated persistent pain. NMDARs directly bind to a family of membrane-associated guanylate kinases (MAGUKs) that regulate surface and synaptic NMDAR trafficking in the CNS. We report here that postsynaptic density-93 protein (PSD-93), a postsynaptic neuronal MAGUK, is expressed abundantly in spinal dorsal horn and forebrain, where it colocalizes and interacts with NMDAR subunits NR2A and NR2B. Targeted disruption of the PSD-93 gene reduces not only surface NR2A and NR2B expression but also NMDAR-mediated excitatory postsynaptic currents and potentials, without affecting surface AMPA receptor expression or its synaptic function, in the regions mentioned above. Furthermore, mice lacking PSD-93 exhibit blunted NMDAR-dependent persistent pain induced by peripheral nerve injury or injection of Complete Freund's Adjuvant, although they display intact nociceptive responsiveness to acute pain. PSD-93 appears to be important for NMDAR synaptic targeting and function and to be a potential biochemical target for the treatment of persistent pain.

Corticosterone alters N-methyl- d-aspartate receptor subunit mRNA expression before puberty

Stress and stress hormones alter the expression of mRNA for the NR1, NR2A and NR2B subunits of the N-methyl- d-aspartate (NMDA) receptor in brain regions associated with the stress response. Early life stress contributes to the risk and pathophysiology of mental illness. Examining how stress hormones modulate NMDA receptor subunit gene expression before and after pubertal onset will further contribute to the understanding of how stress during adolescence relates to adult mental illness. Using in situ hybridization histochemistry, we measured NR1, NR2A and NR2B mRNA expression in the hippocampus and in the hypothalamic paraventricular nucleus (PVN) of rats that had undergone adrenalectomy (ADX) or sham surgery before or after puberty. Some ADX rats received corticosterone pellets that released either normal or stress levels of corticosterone for 14 days prior to sacrifice. There was a significant increase in NR1 subunit mRNA expression throughout the subfields of the hippocampus and in the PVN of ADX prepubertal rats. However, similar changes in hippocampal NR1 expression were not observed in postpubertal ADX rats. Pre- and postpubertal ADX rats implanted with a high-dose corticosterone pellet had decreased expression of PVN NR1 mRNA. Only prepubertal rats had an increase in dentate gyrus NR2A mRNA and CA3 region NR2B mRNA following high-dose replacement. These results provide evidence that glucocorticoids have differential effects on the regional expression of mRNA NMDA receptor subunits and elucidate a window during adolescence in which the NR1, NR2A and NR2B genes are responsive to glucocorticoids.

Dark-rearing decreases NR2A N-methyl- d-aspartate receptor subunit in all visual cortical layers

Maturation of the visual cortex is a visual experience-dependent process. It has been shown that visual input triggers changes in N-methyl- d-aspartate receptor (NMDAR) subunit expression in the visual cortex. However, no data are available on the layer distribution of these molecular changes. Here we describe the laminar distribution of the cells expressing the NMDAR subunits NR2A and NR2B in the rat primary visual cortex at postnatal day (P) 21 and 37 using anti-NR2A and anti-NR2B antibodies and a stereological method to count labelled neurons. The percentage of neurons expressing the NR2A subunit in the layers II–VI remained unchanged between P21 and P37 with a slight decrease in layer V. Dark-rearing from P21 to P37 induced a pronounced decrease of the staining intensity and a significant decrease in the percentage of NR2A-expressing neurons. The changes in NR2A expression caused by dark rearing occur at similar levels in layers II–VI. The percentage of NR2B-positive cells in the different cortical layers remains unchanged from P21 to P37. The NR2B pattern was not significantly affected by dark-rearing. Thusly, the expression of NR2A depends upon visual experience after P21.

Are NMDA receptors in the cortex affected by sensory learning?

NMDA receptor is a heteromeric, ligand‐gated calcium channel, believed to mediate mechanisms of neuronal plasticity. In the adult cerebral cortex the receptor is build up with NR1, NR2A and NR2B subunits. The properties of the receptor are regulated by changing the relative expression of NR2A and NR2B subunits. We have previously found that 3 days of classical conditioning in which stimulation of a row of vibrissae was paired with a tail shock induces NMDA‐receptor dependent changes in cortical representations of the stimulated vibrissae. Expression level of NR2A and NR2B subunits during training was examined by in situ hybridization. NR2A mRNA expression showed a rising tendency in the barrel (layer IV) of the ‘trained’ row of vibrissa after sensory conditioning. At the same time point, NR2A protein level in P2 fraction showed similar increasing tendency. NR2B expression level did not change. The results indicate that a short lasting learning‐dependent task induces a small modification in the composition of NMDA receptor channel complex.

Chronic Prenatal Ethanol Exposure Alters Ionotropic Glutamate Receptor Subunit Protein Levels in the Adult Guinea Pig Cerebral Cortex

Background: The superfamily of glutamate‐gated ion channels mediates fast excitatory synaptic transmission in the central nervous system and is composed of the NMDA, AMPA, and kainate receptors. Binding studies have shown that chronic prenatal and/or neonatal ethanol exposure produces persistent effects on the numbers of some of these channels. However, whether or not this chronic ethanol exposure produces long‐lasting effects on the expression of specific ionotropic receptor subunits remains an open question.Methods: Timed pregnant Dunkin‐Hartley strain guinea pigs received oral administration of one of the following regimens between gestational days 2 and 67: (1) 4 g of ethanol per kilogram of maternal body weight per day with ad libitum access to pellet food and water (ethanol group), (2) isocaloric sucrose‐ and pair‐feeding with ad libitum access to water (sucrose group), or (3) isovolumetric water with ad libitum access to food and water (water group). The maternal blood ethanol concentration produced by the ethanol regimen was 71 ± 12 mM. Adult offspring were killed on postnatal day 61, and cerebral cortical tissue was analyzed for ionotropic glutamate receptor subunit expression by Western immunoblotting.Results: There was a statistically significant decrease in NR2B subunit protein expression and an increase in GluR2/3 subunit protein expression in the ethanol group. Expression of NR1, NR2A, NR2C, GluR1, GluR6/7, and KA2 subunit proteins was not affected.Conclusions: These results demonstrate that chronic prenatal ethanol exposure produces long‐lasting effects on the subunit composition of NMDA and AMPA receptors in the cerebral cortex of the adult guinea pig.

Long-term changes in hippocampal n-methyl- d-aspartate receptor subunits following unilateral vestibular damage in rat

Previous studies have indicated that damage to the peripheral vestibular system results in dysfunction of hippocampal place cells and an impairment of spatial learning and memory. The aim of this study was to determine whether lesions of one vestibular labyrinth (unilateral vestibular deafferentation, UVD) result in changes in the expression of the NR1 and NR2A subunits of the N-methyl- d-aspartate (NMDA) receptor, and the GluR2 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor, in subregions of the rat hippocampus (CA1, CA2/3 and the dentate gyrus) at 10 h or 2 weeks following UVD. Compared with sham surgery controls and anaesthetic controls, the expression of the NR1 subunit was significantly reduced in the ipsilateral CA2/3 region at 2 weeks post-UVD. The expression of the NR2A subunit was also significantly reduced in the ipsilateral CA2/3 and, to a smaller extent, in the contralateral CA2/3 region, at 2 weeks post-UVD. The only other change in NR2A expression was an increase in the ipsilateral CA1 at 10 h post-UVD. No other changes in NR1, NR2A or GluR2 expression were observed in any hippocampal subregion, at any time point, or in cortical tissue at any time point. These results suggest that UVD may result in long-term changes in NMDA receptor subunit expression in the rat hippocampus.

Neuronal gene expression is decreased in prefrontal cortex in schizophrenia

This study aimed to evaluate the effects of repetitive transcranial magnetic stimulation (rTMS) on learning and memory in a rat model of vascular dementia (VaD) and to analyze the associated mechanisms. Bilateral carotid artery occlusion (2-VO) was used to establish a rat model of VaD. High-frequency (5 Hz) rTMS was performed on rats for four weeks. Spatial learning and memory abilities were evaluated using the Morris water maze (MWM), and synaptic plasticity in the hippocampus was assessed via long-term potentiation (LTP). Hippocampal expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF) and three subunits of the N-methyl-d-aspartic acid receptor (NMDAR), NR1, NR2A and NR2B, was analyzed by Western blotting. Compared with the VaD group, escape latency was decreased (P < 0.05) and the time spent in the target quadrant and the percentage of swimming distance within that quadrant were increased (P < 0.05) in the rTMS group. LTP at hippocampal CA3-CA1 synapses was enhanced by rTMS (P < 0.05). VEGF expression was up-regulated following 2-VO and was further increased by rTMS (P < 0.05). BDNF, NR1 and NR2B expression was decreased in the VaD group and increased by rTMS (P < 0.05). There were no significant differences in NR2A expression among the three groups. These results suggest that rTMS improved learning and memory in the VaD model rats via the up-regulation of VEGF, BDNF and NMDARs. In addition, NR2B may be more important than NR2A for LTP induction in the hippocampus during rTMS treatment of VaD.

Expression of NMDA neuroreceptors in experimental ischemia.

The role of NMDA receptors in molecular mechanisms of neurotoxicity was investigated using rat models of global and focal cerebral ischemia. Expression of NR2A and NR2B receptor mRNAs up-regulated in cortex after 3 h of reperfusion following middle cerebral artery occlusion (MCAo). This effect was accompanied by an increase in NR2A and NR2B immunoreactivity. At six hours of reperfusion, drastic activation of NR2A mRNA expression was observed in the penumbra that returned to the control level at 24 h of reperfusion. The monitoring of NR2A autoantibodies in the blood of the experimental rats showed its reliable increase to the 5-6th day of reperfusion that maintained elevated to the 20th day of the experiment. The data indicate that NR2A and 2B receptor subunits and NR2A autoantibodies are biochemical markers of the neurotoxicity underlying cerebral ischemia.

Analysis of Transcriptional Regulatory Sequences of the N-Methyl-d-aspartate Receptor 2A Subunit Gene in Cultured Cortical Neurons and Transgenic Mice

The postnatal appearance and up-regulation of the NR2A subunit of the N-methyl-d-aspartate receptor contributes to the functional heterogeneity of the receptor during development. To elucidate the molecular mechanisms that regulate the neural and developmental specific expression of NR2A, an upstream approximately 9-kb region of the gene harboring the promoter was isolated and characterized in transgenic mice and transfected cortical neurons. Transgenic mouse lines generated with luciferase reporter constructs driven by either 9 or 1 kb of upstream sequence selectively transcribe the transgene in brain, as compared with other non-neural tissues. Reporter luciferase levels in dissociated cultures made from these mice are over 100-fold greater in neuronal/glial co-cultures than in pure glial cultures. Analysis of NR2A 5'-nested deletions in transfected cultures of cortical neurons and glia indicate that while sequences residing upstream of -1079 bp augment NR2A neuronal expression, sequences between -486 and -447 bp are sufficient to maintain neuronal preference. An RE1/NRSE element is not necessary for NR2A neuron specificity. Furthermore, comparison of the 5'-deletion constructs in cortical neurons grown for 5, 8, 11, or 14 days in vitro indicate that sequences between -1253 and -1180 bp are necessary for maturational up-regulation of NR2A. Thus, different cis-acting sequences control the regional and temporal expression of NR2A, implicating distinct regulatory pathways.

NMDA and AMPA receptor subunit protein expression in the rat vestibular nucleus following unilateral labyrinthectomy.

We examined the expression of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor, and the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor, in the ipsilateral and contralateral vestibular nucleus complexes (VNCs) at 10 h and 2 weeks following unilateral vestibular deafferentation (UVD) in rats, in order to directly test the hypothesis that the behavioural recovery following UVD ('vestibular compensation') is associated with an up-regulation of NMDA receptors. We found no significant changes in NR1 or NR2A expression at 10 hs or 2 weeks post-op. compared to sham and anesthetic controls. We did find a significant (p < 0.01 and p < 0.05) increase in GluR2 expression in both VNCs at 10 h but not 2 weeks post-op. compared to sham and anesthetic controls; however, comparison over time post-UVD failed to detect a significant difference, suggesting that it was small and transient at best. These results add further evidence to the conclusion that NMDA receptors do not undergo up-regulation in the ipsilateral VNC during vestibular compensation.

Subunit-Specific NMDA Receptor Trafficking to Synapses

To elucidate mechanisms controlling the number and subunit composition of synaptic NMDA-Rs in hippocampal slice neurons, the NR1, NR2A, and NR2B subunits were optically and electrophysiologically tagged. The NR2 subunit directs delivery of receptors to synapses with different rules controlling NR2A and NR2B. Synaptic incorporation of NR2B-containing receptors is not limited by synaptic transmission nor enhanced by increased subunit expression. NR2A-containing receptors whose expression normally increases with age replace synaptic NR2B-containing receptors. Replacement is enhanced by increased NR2A expression and requires synaptic activity. Surprisingly, spontaneously released transmitter acting on synaptic NMDA-Rs is sufficient for replacement and reduces NMDA-R responses. Thus, as with AMPA-Rs, synaptic trafficking of NMDA-Rs is tightly regulated and has subunit-specific rules with functionally important consequences.

Glutamate-induced currents reveal three functionally distinct NMDA receptor populations in rat dorsal horn – effects of peripheral nerve lesion and inflammation

The importance of the N-methyl- D-aspartate (NMDA) receptor in various painful conditions is well established. The effects of peripheral nerve lesion or joint inflammation, as models of different pain states, on NMDA receptor-mediated currents and NMDA receptor subunit mRNA expression were therefore studied in acutely dissociated neurones from the rat spinal cord dorsal horn. In the neuronal population from control rats, all four NR2 subunits and both NR1 splice variants assayed were detected. A majority of neurones expressed mRNA for more than one NR2 subunit, and some neurones expressed all four NR2 subunits as well as both NR1 splice variants. The NR2B subunit was the most commonly expressed, while the NR2C was the rarest. Following nerve lesion, fewer neurones expressed NR2A compared to the control. The dose–response curve for glutamate-evoked NMDA receptor-mediated currents in the neurones was best described by a three-component fit, suggesting that three functionally distinct NMDA receptor populations are present in the dorsal horn. Minor changes in the dose–response curve after nerve lesion could not be ascribed with certainty to the lesion. Changes in other parameters of NMDA receptor-mediated currents were observed neither after nerve lesion nor after joint inflammation. In summary, the present work demonstrates that single dorsal horn neurones express mRNA for several NMDA receptor subunits. The glutamate dose–response curves indicate that there are three major types of NMDA receptors present in dorsal horn neurones. We also report a reduced expression of NR2A following peripheral nerve lesion.

Further characterization of the molecular interaction between PSD-95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating.

Coexpression of PSD-95(c-Myc) with NR1-1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3 H]MK801 binding similar to that previously described for l-glutamate. The inhibition constants (K (I) s) for the binding of l-glutamate and glycine to NR1-1a/NR2A determined by [3 H]CGP 39653 and [3 H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1-1a/NR2A receptors coexpressed +/- PSD-95(c-Myc). The increased EC(50) for l-glutamate enhancement of [3 H]MK801 binding was also found for NR1-2a/NR2A and NR1-4b/NRA receptors thus the altered EC(50) is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1-4b but not the NR1-1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1-4b and NR1-4b/NR2A expression was enhanced by PSD-95(c-Myc) but whole cell enzyme-linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD-95(c-Myc) has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C-terminal E(T/S)XV PSD-95 binding motifs.

Developmental expression of NMDA receptors in primary cortical cell culture: relationship to function and excitotoxicity

Over‐activation of the N‐methyl‐d‐aspartate (NMDA) receptor results in a Ca2+‐dependent neurotoxicity termed excitotoxicity. Primary neuronal cell cultures are often used to study the mechanisms of excitotoxicity. While the expression of the NMDA receptor (NR) subunits and their relationship to Ca2+ entry/accumulation and excitotoxicity has been studied extensively, all three parameters have not been examined concurrently. To determine unequivocally whether developmental expression of NR protein and function do indeed coincide with the appearance of excitotoxicity, we examined the temporal relationship between NR subunit expression, NMDA‐induced Ca2+ accumulation, and NMDA‐mediated excitotoxicity simultaneously using sister plates derived from the same mixed cortical cell culture preparations. Western Blot analysis of total protein isolated from cells cultured for 1, 4, 7, 10 and 14 days revealed a time‐dependent increase in NR1, NR2A and NR2B subunit expression, which surprisingly did not correlate with NMDA receptor function, as assessed by measurement of NMDA‐induced 45Ca2+ accumulation. However, when only NR subunit surface expression was quantified, a correlation between expression and 45Ca2+ accumulation did indeed exist. To our surprise, the emergence of excitotoxicity did not show a direct relationship to 45Ca2+ accumulation as has been reported previously. Thus, it appears that other factors besides total Ca2+ accumulation must contribute to the emergence of excitotoxicity in mixed murine cortical cell cultures.Acknowledgements: Supported by a grant from The Patrick and Catherine Weldon Donaghue Medical Foundation.

Differential NR2A and NR2B expression between trigeminal neurons during early postnatal development

N-methyl-D-aspartate (NMDA) receptors play an important role in the production of rhythmical trigeminal motor activity resembling suckling and chewing. The developmental relationship between the expression of NMDA receptor subunits and the function of neurons comprising brainstem oral–motor circuitry is not clear. We conducted receptor immunohistochemistry studies to demonstrate the expression of NR2A and NR2B subunits in trigeminal motoneurons (Mo5) and mesencephalic trigeminal neurons (Me5) during the first 2 weeks of development. During this time period, rats begin the transition from suckling to chewing, two distinct motor behaviors. In Mo5, NR2A and NR2B immunoreactivity was observed throughout the time frame sampled. A significant increase in the NR2A:NR2B ratio occurred between P3–4 and P11 due to a reduction in the number of NR2B immunoreactive neurons. The temporal and spatial expression of NR2A and NR2B was differentially regulated between caudal and rostral regions of Me5. In contrast to Mo5, the NR2A:NR2B ratio decreased between P0–1 and P11 in caudal Me5 due to a concurrent increase in the number of NR2A and NR2B immunoreactive neurons. In rostral Me5, NR2A and NR2B immunoreactivity emerged at P3 and P11, respectively. Our data provides further insight into the molecular changes of trigeminal neurons during the transition from suckling to chewing behaviors. The differences in the NR2A:NR2B ratio between Mo5 and Me5 suggest functional differences in these neurons during NMDA-mediated neurotransmission. Synapse 44:76–85, 2002. © 2002 Wiley-Liss, Inc.

Postnatal development of NR2A and NR2B mRNA expression in rat auditory cortex and thalamus.

Sensory cortex in the rat undergoes rapid postnatal development, especially following the onset of sensory function during so-called "critical periods." To investigate potential mechanisms in the auditory forebrain involving different NMDA receptor subunits, we have used in situ hybridization to determine expression patterns of NR2A and NR2B mRNA at postnatal days 4, 10, 13, 18, 25, and adult. In auditory cortex, NR2A mRNA expression is initially weak but increases rapidly over approximately 2 weeks. NR2B mRNA levels are initially high and remain high. For both subunits, expression tends to be highest in superficial layers of the cortex (except layer 1). Expression is weaker in the auditory thalamus (medial geniculate). Initially, NR2A mRNA expression is very low, whereas NR2B mRNA expression is moderate; both levels increase over approximately 2 weeks. Among medial geniculate subdivisions, NR2A mRNA expression occurs preferentially in the medial division, whereas NR2B mRNA expression is strongest in the ventral division. For auditory cortex and thalamus, NR2A and NR2B mRNA expression peaks about 1 week after the onset of hearing before declining slightly into adulthood. The heterogeneous distribution of NMDAR subunit mRNA throughout development may play a role in auditory forebrain development and function.

Reduced NR2A expression and prolonged decay of NMDA receptor-mediated synaptic current in rat vagal motoneurons following axotomy.

To elucidate characteristic changes in the N-methyl-D-aspartate (NMDA) receptor on neurons following axotomy, subunit expressions and functional features of the NMDA receptor were examined in the dorsal motor nucleus of vagus (DMV) of rats receiving vagal axotomy at the neck. Western blotting analysis demonstrated that the expression of NR2A decreased 2-3 days after in vivo axotomy, while expression of NR1 and NR2B, NR2C and NR2D subunits did not change significantly. To examine the functional changes, patch clamp recordings in whole-cell mode were employed on the axotomized DMV neurons identified by retrograde labelling with fluorescent dye. The amplitude ratios of ifenprodil-sensitive components of NMDA response and D,L-2-amino-5-phosphovaleric acid (APV)-sensitive evoked postsynaptic current increased after axotomy. In addition, APV-sensitive postsynaptic currents exhibited a longer decay time in identified axotomized vagal motoneurons than in control neurons. No significant differences in the current density of the NMDA response and the peak amplitude of APV-sensitive synaptic currents were observed between axotomized and intact DMV neurons. In conclusion, a decrease in NR2A expression results in the appearance of functional characteristics of the NMDA receptor predominantly containing the NR2B subunit. This might lead to a long-term increase of the susceptibility of neurons to excitotoxicity.