NR1 Expression Research Articles

Human T lymphocytes express N-methyl- d-aspartate receptors functionally active in controlling T cell activation

The aim of this study was to investigate the expression and the functional role of N-methyl- d-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 μg/ml) treatment. d-(−)-2-Amino-5-phosphonopentanoic acid ( d-AP5), and (+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.

RNA interference of Xenopus NMDAR NR1 in vitro and in vivo

Here, we present a short interfering RNA (siRNA) application that reduces the expression of NR1 the obligate subunit of the NMDA receptor (NMDAR) and virtually eliminates NMDAR function in a small subset of neurons within otherwise normally developing Xenopus laevis tadpoles. We designed two plasmids each containing a CMV promoter driving a dsRed “reporter” cDNA and DNA coding for one short hairpin RNA (shRNA) under the control of the U6 promoter. The shRNA was cleaved to produce a siRNA against NR1 transcript (iNR1). NR1 transcript and protein, differentiation and survival of NR1 knockdown neurons, were assayed in vivo. iNR1 effects on NMDAR function used Xenopus tectal neuron cultures, Ca 2+ imaging and patch-clamp electrophysiology. NR1 transcript, protein and NMDAR function was significantly reduced or eliminated in iNR1-expressing neurons. Protein, transcript and function for the closely related AMPA receptor (AMPAR) subunit GluR1 was unperturbed. In vivo imaging of small groups of iNR1 neurons at two different ages revealed that siRNA knockdown of the NMDAR does not affect differentiation or survival of young neurons. Thus, with siRNA, molecular hypotheses about synaptic mechanisms of circuit formation can be rapidly tested in intact developing vertebrates using normal neurons in the same animals as controls.

NMDA receptor antagonist treatment at the time of nerve injury prevents injury-induced changes in spinal NR1 and NR2B subunit expression and increases the sensitivity of residual pain behaviours to subsequently administered NMDA receptor antagonists

Spinal NMDA receptors (NMDA R) are important in neuropathic sensitisation and acute administration of antagonists can provide temporary attenuation of sensitisation. If establishment of the chronic pain state could be prevented by brief administration of such agents at or around the time of nerve injury (pre-emptive analgesia) it might be possible to avoid many of the unacceptable side effects associated with repeated administration of these or other antagonists. Several reports describe aspects of effective pre-emptive analgesia from NMDA R antagonists in animal models of neuropathic pain. The first aim of the present study was to make a direct comparison of changes in mechanical allodynia, cold allodynia and thermal hyperalgesia following nerve injury, demonstrating their increasing degree of susceptibility to pre-emptive NMDA R antagonist treatment. Secondly, we used immunoblotting and immunohistochemistry to investigate the effects of nerve injury on NMDA receptor subunit expression, revealing increased expression of NR2B, but not NR2A and reduced NR1 in the superficial dorsal horn. These changes were attenuated following NMDA receptor antagonist pre-treatment. Thirdly, we investigated the pharmacological properties of residual mechanical allodynia and cold allodynia that remained after pre-emptive treatment and revealed a greater sensitivity to NMDA R antagonists. These findings indicate that in addition to a marked suppression of thermal hyperalgesia and cold allodynia, pre-emptive treatment with NMDA R antagonist causes a lasting change in spinal NMDA R complexes such that remaining mechanical allodynia should be more effectively targeted by NMDA R antagonists.

NMDA receptor subunit NRI and postsynaptic protein PSD-95 in hippocampus and orbitofrontal cortex in schizophrenia and mood disorder

Much interest has focussed on glutamate and the N-Methyl-D-Aspartate (NMDA) glutamate receptor in the pathogenesis of schizophrenia. A number of studies have reported abnormal gene transcription of various glutamate receptor subtypes in the hippocampus including the NMDA receptor. However, corresponding protein levels in subregions of the hippocampus have not yet been investigated. We have used immunoautoradiographical techniques to assess the expression of the obligatory NMDA receptor subunit NR1 and an associated post-synaptic density protein PSD-95 in the hippocampal dentate gyrus and orbitofrontal cortex (OFC) in schizophrenia and mood disorder. Optical density measures from film autoradiographs revealed no changes in NR1 or PSD-95 in the OFC or dentate hilus, however a decrease in PSD-95 was found in the dentate molecular layer in both schizophrenia and bipolar disorder relative to major depression. These findings were unrelated to antipsychotic or mood stabilizer drug treatment. The dentate molecular layer contains the dendritic trees of granule cells and is the target of major excitatory afferent inputs from associative cortical, parahippocampal and hippocampal regions. A reduction in PSD-95 at glutamate synapses of the molecular layer may have a deleterious impact on information flow to other hippocampal regions via granule cells and their projecting mossy fibres. A down-regulation of PSD-95 in schizophrenia and bipolar disorder may also relate to disease mechanisms of psychosis.

NR1, NR2A and NR2C subunits expression after cervical spinal cord transplant and section in dogs

This paper served to evaluate the expression levels of subunits NR1, NR2A and NR2C which are implicated in neuronal plasticity events. A 50% (right half) 4 mm longitudinal resection of the spinal cord was done at the C5–C6 level with preservation of the anterior spinal artery. This was effected in a dog model after either a homologous transplant or a pure spinal cord section. In this study we used two groups of dogs with four individuals each, as well as a control group. The transplant group ( n = 4) was analyzed at days 3 and 28 post surgery. The section group ( n = 4) was also analyzed at days 3 and 28 post op. All three groups (transplant, section and control) were evaluated as to the subunit expression in each of the segments corresponding to the transplanted or sectioned sites, the site contralateral to the transplanted or sectioned sites at levels half a centimeter both proximal and distal to the site of transplant and section. The results showed a variety of changes in each of the subunits depending on the group, the segment and the time of evaluation (acute versus chronic). This could be closely related to mechanisms which participate in regeneration and functional recuperation.

The Metabotropic Glutamate 5 Receptor Antagonist 3-[(2-Methyl-1,3-thiazol-4-yl)ethynyl]-pyridine Reduces Ethanol Self-Administration in Multiple Strains of Alcohol-Preferring Rats and Regulates Olfactory Glutamatergic Systems

The metabotropic glutamate 5 receptor (mGlu5) receptor has been implicated as having a role in pain modulation, anxiety, and depression, as well as drug-seeking behavior. In the present study, we examined the effect of the selective mGlu5 receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) on operant ethanol self-administration by two strains of rats, the Fawn-Hooded (FH) rat and the inbred alcohol-preferring (iP) rat. MTEP (2 mg/kg i.p.) caused a significant reduction in responding for ethanol by both strains of rats; however, in the iP rats, MTEP also induced apparent sedation at this dose, although still reduced alcohol responding at lower doses. Chronic MTEP (2 mg/kg/day) caused a significant reduction in ethanol consumption by FH rats in a two-bottle preference test; however, chronic treatment with this dose had no effect on anxiety-like behavior or depressive-like behavior in FH rats, suggesting the dose used was subthreshold for anxiolytic or antidepressive-like effects. Finally, repeated dosing with MTEP (2 mg/kg i.p.) caused significant reductions in expression of the mRNA encoding the NR1 subunit of the N-methyl-D-aspartate receptor and the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor in the cingulate cortex. A significant decrease in NR1 expression also occurred in the piriform cortex. Chronic MTEP also caused a significant decrease in mGlu5 gene expression and a significant increase in dopamine transporter and dopamine D(2)-like receptor binding within the olfactory tubercle. Collectively, these data suggest that MTEP can reduce alcohol-seeking behavior in different rodent models of alcoholism, and this effect is associated with regulation of cortical glutamate systems, particularly those in olfactory-related regions.

Down-regulation of N-methyl D-aspartate receptor in rat-modeled disuse osteopenia

Lack of mechanical stress may result in osteoporosis; however, the underlying mechanisms of disuse osteoporosis remain unclear. It has been indicated that mechanical loading causes extracellular glutamate accumulation in osteoblasts. We hypothesized that the glutamate receptor mediation on bone cells might also be involved in mechanically stimulated osteogenesis. In this study, we investigated the changes of bone formation and the expressions of osteogenic genes and N-methyl D-aspartate (NMDA) receptors, the major glutamate receptors, in disused bones. Rat modeled disuse osteopenia in hind limbs was induced by a 3-week tail suspension in Sprague-Dawley rats. Bone mineral density and trabecular bone volume of distal femurs were measured to verify the osteopenia of disused bones. The mRNA expressions of cbfa1/Runx2, type I collagen, alkaline phosphatase (ALP) and osteocalcin (OC) in bones were measured as osteogenic markers. The influences of mechanical unloading on the expressions of NMDA receptors (NR1 and NR2D) in bones were also examined. The effects of NMDA mediation on osteogenesis were tested by a treatment of MK-801, a non-competitive NMDA receptor antagonist, in cultured osteoblasts and bone marrow stroma cells. Our result showed that mRNA expressions of cbfa1/Runx2, type I collagen, ALP and OC were significantly decreased in disused bones. The mRNA and protein expressions of NR1 and NR2D were significantly decreased in disused bones; furthermore, immunolocalization of both receptors showed decreases in osteoblasts, but not in osteoclasts. The results from the in vitro study showed that MK-801 inhibited mRNA expression of cbfa1/Runx2 in bone marrow stroma cells and also inhibited those of collagen type I, ALP and OC of osteoblasts in a dose-dependent manner. These results suggest that NMDA receptor mediation may play an important role in transmitting mechanical loading in bones, and decreases of the expressions of NMDA receptors in disused bones, especially in osteoblasts, may contribute to the decrease of osteogenesis.

Maternal zinc deficiency reduces NMDA receptor expression in neonatal rat brain, which persists into early adulthood

Prenatal and early postnatal zinc deficiency impairs learning and memory and these deficits persist into adulthood. A key modulator in this process may be the NMDA receptor; however, effects of zinc deficiency on the regulation of NMDA receptor activity are not well understood. Female Sprague-Dawley rats were fed diets containing 7 (zinc deficient, ZD), 10 (marginally zinc deficient, MZD) or 25 (control) mg Zn/g diet preconception through postnatal day (PN) 20, at which time pups were weaned onto their maternal or control diet. Regulation of NMDA receptor expression was examined at PN2, PN11, and PN65. At PN2, expression of whole brain NMDA receptor subunits NR1, NR2A, and NR2B was lower in pups from dams fed ZD and MZD compared to controls, as analyzed using relative RT-PCR and immunoblotting. At PN11, whole brain and hippocampi NR1, NR2A, NR2B and PSA-NCAM (polysialic acid-neural cell adhesion molecule) expression and the number of PSA-NCAM immunoreactive cells were lower in pups from dams fed ZD compared to controls. Whole brain brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) concentrations were lower in pups from dams fed ZD or both low zinc diets, respectively. Whole brain NR1 expression remained lower in previously zinc-deficient rats at PN65. These data indicate potential mechanisms through which developmental zinc deficiency can impair learning and memory later in life.

An Opiate Cocktail that Reduces Morphine Tolerance and Dependence

Morphine is an exceptionally effective analgesic whose utility is compromised by the development of tolerance and dependence to the drug. Morphine analgesia and dependence are mediated by its activity at the mu opioid peptide (MOP) receptor [1]. The MOP receptor is activated not only by morphine, but also by other opiate drugs such as methadone and endogenous opioids such as endorphins. Morphine, however, is a unique opioid agonist ligand because it fails to induce endocytic trafficking of the MOP receptor [2], whereas the endogenous ligands and methadone do facilitate endocytosis [3]. Using the unique pharmacology of the MOP receptor and its proposed existence as an oligomeric structure [4], we designed a pharmacological cocktail that facilitates endocytosis of the MOP receptor in response to morphine. This cocktail consists of morphine and a small dose of methadone. Importantly, this cocktail, while retaining full analgesic potency, does not promote morphine dependence. We further demonstrate that dependence is reduced, at least in part, because endocytosis of the MOP receptor in response to morphine prevents the upregulation of N-methyl-D-aspartate (NMDA) receptors.

Activation of the ERK1/2 signaling cascade by excitotoxic spinal cord injury

The role of the ERK1/2 signal transduction pathway and related transcription factors in the regulation of gene expression and pain behavior following excitotoxic spinal cord injury (SCI) was examined. Specifically, phosphorylation of ERK1/2, activation of transcription factors NF-kB, ELK-1, and CREB, and gene expression of the neurokinin-1 receptor and NMDA receptor subunits NR1 and NR-2A were investigated. Excitotoxic injury was produced by intraspinal injection of quisqualic acid (QUIS) in male Sprague–Dawley rats. Western blots were used to evaluate phosphorylation and activation of ERK1/2 and transcription factors using phospho-specific or total antibodies. Real-time PCR was used to evaluate gene expression of NK-1R, NR-1, and NR-2A. Assessment of excessive grooming behavior was used to evaluate the presence of spontaneous pain behavior. Excitotoxic spinal injury resulted in: (1) increased phosphorylation of ERK1/2; (2) increased activation of NF-kB and phosphorylation of ELK-1; and (3) increased gene expression for the NK-1 receptor and NR1 and NR-2A subunits of the NMDA receptor. Blockade of the ERK cascade with the MEK inhibitor PD98059 inhibited phosphorylation of ELK-1, activation of NF-kB and gene expression of NR1, NR-2A and NK-1R, and prevented the development of excessive grooming behavior. The results have shown that excitotoxic spinal injury leads to the injury-induced activation of the ERK→ELK-1 and NF-kB signaling cascades and transcriptional regulation of receptors important in the development of chronic pain. Blockade of this intracellular kinase cascade prevented the onset of injury-induced pain behavior.

Protein phosphatase modulates the phosphorylation of spinal cord NMDA receptors in rats following intradermal injection of capsaicin

The present study investigates the role of serine/threonine protein phosphatase 2A (PP2A) in the modulation of the phosphorylation of the NR1 and NR2B subunits of NMDA receptors in the spinal cord of rats following intradermal injection of capsaicin. The effects of a specific inhibitor of PP2A, fostriecin, on the expression of NR1, phospho-NR1, NR2B, and phospho-NR2B subunits of the NMDA receptor in the spinal cord of rats following noxious stimulation were examined. After continually perfusing with ACSF or fostriecin (3 μM) through a previously implanted microdialysis fiber for 30 min, central sensitization was initiated by injection of capsaicin into the plantar surface of the left paw of rats. The spinal cord was removed at different time points (30, 60, 90, 120, 180 min) after intradermal injection of capsaicin. Western blots were performed to examine the expression of NMDA subunits in spinal cord tissue by using specific antibodies. We found that the upregulated phosphorylation of both NR1 and NR2B subunits induced by capsaicin injection was significantly potentiated by the PP2A inhibitor without affecting the NR1 and NR2B protein itself. These results suggest that PP2A may have a regulatory effect on central sensitization induced by noxious stimuli in the periphery by regulating the phosphorylation state of NMDA receptors.

Identification of a homocysteine receptor in the peripheral endothelium and its role in proliferation

Homocysteine, a risk factor for atherosclerosis, increases intimal hyperplasia after carotid endarterectomy with associated smooth muscle cell proliferation and modulation of cytokines. The N-methyl-D-aspartate receptor (NMDAr), a glutamate-gated ion channel receptor, is associated with homocysteine-induced cerebrovascular injury; however, the receptor has not been identified in peripheral vascular cells, nor has any interaction with homocysteine been clarified. Our objectives were first, to identify NMDAr in rat carotid artery and rat aorta endothelial cells (RAEC); and second, to determine whether homocysteine activates NMDAr in the endothelium. NR1 and NR2A, two NMDAr subunits, were probed in rat carotid arteries by immunohistochemistry. RNA was isolated from RAECs, and expression of all NMDAr subunits (NR1, 2A, 2B, 2C, and 2D) were examined by RT-PCR and sequencing. For receptor protein expression, RAEC were incubated with different homocysteine concentrations and incubation times and also were treated with 50 microM homocysteine and/or preincubated with 50 microM dizocilpine MK-801, an NMDAr inhibitor. Both NR1 and NR2A were expressed in rat carotid arteries. All NMDAr subunits were expressed in the RAECs, and there was 92% to 100% similarity compared with rat NMDAr from the National Center for Biotechnology Information (NCBI) GenBank. Homocysteine upregulated NR1 expression and increased cell proliferation. RAEC pretreatment with MK-801 reduced homocysteine-mediated cell proliferation. This study is the first to show that NMDAr exists in the peripheral vasculature, and that homocysteine may act via NMDAr to increase intimal hyperplasia. Our objectives included the identification of a homocysteine receptor in the peripheral vasculature. The possible inhibition of a homocysteine receptor to prevent intimal hyperplasia rather than treat established stenosis would make a significant clinical impact. This will open further avenues of study in determining the role of homocysteine in the pathogenesis of intimal hyperplasia.

Counteraction by repetitive daily exposure to static magnetism against sustained blockade of N‐methyl‐D‐aspartate receptor channels in cultured rat hippocampal neurons

In rat hippocampal neurons cultured with the antagonist for N-methyl-D-aspartate (NMDA) receptors dizocilpine (MK-801) for 8 days in vitro (DIV), a significant decrease was seen in the expression of microtubule-associated protein-2 (MAP-2) as well as mRNA for both brain-derived neurotrophic factor (BDNF) and growth-associated protein-43 (GAP-43), in addition to decreased viability. MK-801 not only decreased the expression of the NR1 subunit of NMDA receptors but also increased NR2A expression, without affecting NR2B expression. Repetitive daily exposure to static magnetic fields at 100 mT for 15 min led to a decrease in the expression of MAP-2, without significantly affecting cell viability or the expression of neuronal nuclei (NeuN) and GAP-43. However, the repetitive magnetism prevented decreases in both BDNF mRNA and MAP-2 and additionally increased the expression of NR2A subunit, without altering NR1 expression in neurons cultured in the presence of MK-801. Repetitive magnetism was also effective in preventing the decrease by MK-801 in the ability of NMDA to increase intracellular free Ca2+ ions, without affecting the decrease in the maximal response. These results suggest that repetitive magnetism may at least in part counteract the neurotoxicity of MK-801 through modulation of the expression of particular NMDA receptor subunits in cultured rat hippocampal neurons.

Spinal Cord NR1 Serine Phosphorylation and NR2B Subunit Suppression following Peripheral Inflammation

BackgroundSpinal cord N-methyl-D-aspartate (NMDA) receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury.ResultsLumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation.ConclusionThese data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease.

Spinal Axonal Injury Induces Brief Downregulation of Ionotropic Glutamate Receptors and No Stripping of Synapses in Cord-Projection Central Neurons

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.

Spinal Axonal Injury Induces Brief Downregulation of Ionotropic Glutamate Receptors and No Stripping of Synapses in Cord-Projection Central Neurons

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.

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons.

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.

Long-term effects of neonatal seizures on subsequent N-methyl- d-aspartate receptor-1 and gamma-aminobutyric acid receptor A-α1 receptor expression in hippocampus of the Wistar rat

To evaluate the pathophysiological mechanism of subsequent reduced seizure threshold following neonatal seizures, single or recurrent prolonged seizures were induced to neonatal rats by the inhalant flurothyl. The expression of N-methyl- d-aspartate receptor 1 (NR1) and gamma-aminobutyric acid receptor A-α1 (GABA-A-α1) immunoreactivity in hippocampus were examined by Western blotting analysis at the day 7 (P7) and day 75 (P75) after the last seizure. Whereas there were no significant changes in single seizure group and recurrent seizure group of P75, NR1 expression enhanced significantly in P7 rats of recurrent seizure group. Meanwhile, polypeptide levels of GABA-A-α1 receptor subunit decreased significantly in both single and recurrent seizure-treated P7 and P75 rats. Our results suggest that recurrent or single prolonged seizures during the neonatal period may have long-term effects on the balance between excitatory NMDA system and inhibitory GABA system in hippocampus of rats.

Long-term effects of seizures in neonatal rats on spatial learning ability and N-methyl- d-aspartate receptor expression in the brain

For the purpose of investigating the long-term effects of seizures in neonatal rats on spatial learning ability and N-methyl- d-aspartate (NMDA) receptor expression in adult rat brain, a seizure was induced by inhalant flurothyl daily in neonatal Wistar rats from postnatal day 6 (P6). The authors assigned six rats each averagely into the single-seizure group, the recurrent-seizure group (seizures induced in six consecutive days), and the control group. During P60 to P65, the rats were tested for spatial learning ability with the Morris water maze task. On P75, the authors examined protein expression of the NMDA receptor (NR) subunits, NR1, 2A, 2B, 2C, and 2D, in the cerebral cortex and hippocampus by Western blotting analysis. On P65, the escape latencies from the water maze of the rats in the recurrent-seizure group were significantly longer than those of the control rats, but there was no difference between the single-seizure group and the control group. NR subunit expression in the cerebral cortex and hippocampus of the rats with single seizure was similar to those in the control rats. Compared with the control rats, the protein expressions of NR1, NR2A and NR2B in the cerebral cortex and NR2A in the hippocampus of the recurrent-seizure group was significantly decreased, but NR2C protein expression in the cerebral cortex and hippocampus significantly increased. Recurrent seizures induced in neonatal rats might cause long-term spatial learning ability deficit and modify NR expression in the cerebral cortex and hippocampus of adult rats. The results suggest that abnormal NR expression might play an important role in long-term spatial learning ability deficit induced by recurrent seizures in early life.

Regulation of N-methyl-D-aspartate receptor subunit expression in the fetal guinea pig brain.

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.