Hippocampal expression of N-methyl-D-aspartate receptor (NMDAR1) subunit splice variant mRNA is altered by developmental exposure to Pb(2+)

N-methyl-D-aspartate receptors (NMDAR) play an important role in synaptic plasticity and brain development. We have previously shown that NR1-pan mRNA is significantly increased in the hippocampus of rats chronically exposed to low levels of lead (Pb(2+)) during development [T.R. Guilarte, J.L. McGlothan, Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure, Brain Res., 790 (1998) 98-107]. It is not known whether this Pb(2+)-induced increase in NR1-pan mRNA is associated with changes in specific splice isoforms. To study this effect, we used in situ hybridization of oligonucleotides to probe for the NR1-a, NR1-b, NR1-1, NR1-2, and NR1-4 isoforms which are most abundantly expressed in the rat hippocampus. Developmental exposure to increasing levels of Pb(2+) resulted in significant increases in NR1-a mRNA throughout the pyramidal and granule cell layers of the rat hippocampus at postnatal day 14 (PN14). NR1-b mRNA was increased in the pyramidal cell layer of Pb(2+)-exposed rats at PN21. Splicing of the C-terminus cassettes was also regulated by developmental exposure to Pb(2+). NR1-2 mRNA was increased in CA4 pyramidal cells and in dentate granule cells of PN21 Pb(2+)-exposed rats. Notably, expression of NR1-4 mRNA in CA3 pyramidal cells was increased in Pb(2+)-exposed rats at PN14 and decreased at PN21. No significant Pb(2+) effect was measured for NR1-1 mRNA expression. These data indicate that alternative splicing of the NR1 gene shows selective anatomical and temporal regulation by Pb(2+) in the developing rat hippocampus. This study provides further support to the hypothesis that NMDARs are important targets for Pb(2+)-induced neurotoxicity.     

NMDAR-2A subunit protein expression is reduced in the hippocampus of rats exposed to Pb2+ during development

Chronic exposure to lead (Pb2+) produces deficits of learning and memory in children and spatial learning deficits in developing rats. The N-methyl-D-aspartate receptor (NMDAR) has been identified as a principal target for Pb2+-induced neurotoxicity. Age-dependent changes in NMDAR subunit gene expression were observed in hippocampi of rats chronically exposed to Pb2+ during development [T.R. Guilarte, J.L. McGlothan, Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure, Brain Res. 790 (1998) 98-107]. These changes were present at blood Pb2+ levels ranging from 20-60 microg/dl. Littermates were used in the present study to determine whether the changes in gene expression were reflected in protein levels. NR1, NR2A, and NR2B subunit protein levels were measured in rat hippocampus and cortex at post-natal days (PND) 7, 14, 21, and 28 by Western blot and densitometric analysis. A treatment effect was apparent for NR2A subunit protein expression in the hippocampus (F1,28=10.224, p<0.01). NR2A subunit protein was reduced by 40%, 19%, and 27% from control levels in PND14, 21, and 28 Pb2+-exposed rats, respectively. Mean comparisons indicated that rats at PND14 exhibited the most significant reduction of NR2A (p<0.001). These data concur with our previous finding of reduced NR2A mRNA found in hippocampal pyramidal and granule cells of Pb2+-exposed rats. Pb2+ exposure during development had no effect on NR1 or NR2B subunit protein expression in the hippocampus at any age. No effect was observed on any subunit in the cortex at any age. The developmental profile of the NMDAR-2A subunit protein in the hippocampus is specifically changed by chronic exposure to Pb2+. These data suggest that composition of subunits comprising NMDAR may be altered in Pb2+-exposed rats.     

Effects of chronic alcohol consumption on the expression of different NR1 splice variants in the brain of AA and ANA lines of rats.

Following chronic alcohol treatment alterations in N-methyl-D-aspartate receptor subunit 1 and 2 (NR1 and NR2), mRNA and protein levels have been reported. The NR1 gene undergoes alternative RNA splicing, resulting in eight splice variants, which were shown to differ in their sensitivity to alcohol. Here, we studied mRNA and protein levels of NR1 splice variants in alcohol-preferring (AA) and alcohol-nonpreferring (ANA) rat lines under basal conditions (alcohol-naive), and following chronic alcohol consumption. mRNA levels of three NR1 splice variants (NR1-1, NR1-2, NR1-4), and the protein levels of NR1 (NR1-1/NR1-2), and of NR1 alternative C-terminus (NR1-3/NR1-4) were determined in the hippocampus and nucleus accumbens by competitive RT-PCR and Western blot analysis, respectively. No significant differences in NR1 mRNA, or protein levels were found in the nucleus accumbens between the two rat lines under basal conditions, or following chronic alcohol consumption. In the hippocampus of alcohol-naive rats, the NR1-4 mRNA content was significantly higher in ANA compared to AA rats, however, no significant difference could be detected at the protein level. Following chronic alcohol consumption, the protein level of the NR1 alternative C-terminus (NR1-3/NR1-4) was significantly higher in AA rats compared to the corresponding control. Taken together, these results suggest: (i) brain site-specific alterations in NMDA receptor subunit composition occur following chronic alcohol consumption. (ii) In the hippocampus, NR1 splice variant mRNA levels differ between AA and ANA rats. (iii) The mRNA levels and protein levels of NR1 splice variants are differentially affected by chronic alcohol consumption.     

Developmental Pb2+ exposure alters NMDAR subtypes and reduces CREB phosphorylation in the rat brain

In the present study we show that chronic exposure to low levels of lead (Pb(2+)) during development alters the type of N-methyl-D-aspartate receptor (NMDAR) expressed in the developing and young adult rat brain. Ifenprodil inhibition of [3H]MK-801 binding to the NMDAR channel in cortical and hippocampal neuronal membranes expressed high and low affinity components. Previous studies have shown that the high affinity component is associated with NR1/NR2B receptor complexes while the low affinity component is associated with the appearance and insertion of the NR2A subunit to NMDAR complexes. Pb(2+)-exposed rats express a greater number of [3H]MK-801 binding sites associated with the high affinity and low affinity components of ifenprodil inhibition. Further, [3H]ifenprodil saturation isotherms and Scatchard analysis in cortical and hippocampal membranes showed a higher number of binding sites (B(max)) with no change in binding affinity (K(d)) in Pb(2+)-exposed animals relative to controls. Quantitative [3H]MK-801 autoradiography in response to glutamate and glycine provided evidence that NMDAR complexes in Pb(2+)-exposed rat brain were maximally activated in situ. Higher levels of ifenprodil-sensitive binding sites and increased sensitivity to agonists are properties characteristic of NR1/NR2B recombinant receptors. Thus, our results strongly suggest that a greater proportion of the total number of NMDAR are NR1/NR2B receptors in the Pb(2+)-exposed rat brain. This Pb(2+)-induced change in NMDAR subtypes in the rat brain was associated with reduced CREB phosphorylation in cortical and hippocampal nuclear extracts. These findings demonstrate that chronic exposure to environmentally relevant levels of Pb(2+) altered the subunit composition of NMDAR complexes with subsequent effects on calcium-sensitive signaling pathways involved in CREB phosphorylation.     

Hippocampal NMDA receptor mRNA undergoes subunit specific changes during developmental lead exposure

The N-methyl- -aspartate (NMDA) receptor has shown to play an important role in the cognitive deficits associated with developmental lead (Pb) exposure. In this study, we examined the effects of low-level Pb exposure on NMDA receptor subunit gene expression in the developing rat brain. The pattern of NR1, NR2A, NR2B, and NR2C subunit mRNA in situ hybridization was consistent with previous studies. Brain levels of NR1 and NR2A mRNAs were lowest shortly after birth, increasing to reach peak levels by 14 or 21 days of age and subsequently decreasing at 28 days of age. NR2B mRNA levels were highest during early development and decreased as the animals aged. NR2C subunit mRNA was restricted to the cerebellum and a signal was not detectable until the second week of life. Lead exposure resulted in significant and opposite effects in NR1 and NR2A subunit mRNA expression with no changes in NR2B or NR2C subunit expression. The Pb-induced changes in NR1 and NR2A subunit mRNA were mainly present in the hippocampus. Hippocampal NR1 mRNA levels were significantly increased in the CA1 (15.3%) and CA4 (26.8%) pyramidal cells from 14-day-old Pb-exposed rats. At 21 days of age, only the NR1 mRNA at the CA4 subfield remained significantly elevated (10.3%). Lead exposure caused reductions of NR2A mRNA levels (11.9–19.3%) in the pyramidal and granule cell layers of the hippocampus at 14 and 21 days of age. NR1 mRNA levels were also significantly increased (14.0%) in the cerebellum of 28-day-old rats with no change in NR2A mRNA at any age. No significant changes in subunit mRNA levels were present in cortical or subcortical regions at any age. The Pb-induced changes in hippocampal NMDA receptor subunit mRNA expression measured in the present study may lead to modifications in receptor levels or subtypes and alter the development of defined neuronal connections which require NMDA receptor activation.     

Lack of functional expression of NMDA receptors in PC12 cells

PC12 cells are an established model for studying the role of N-methyl-d-aspartate (NMDA) receptors in excitotoxicity and function as multimeric assemblies of NR1 with at least one NR2(A-D) subunit. We examined NR1 splice variant and NR2 subunit expression in four PC12 cell-lines (ATCC, WEHI, Ordway and Flinders), correlated mRNA expression with protein expression, and used patch-clamp recordings to test functionality. PCR indicated strong expression of the NR1 splice variants NR1-2a and NR1-4a in all cell-lines, with the remainder weakly detected or absent. Real-time PCR showed variable levels of NR1 mRNA expression (all splice variants) between cell-lines and a significant increase in response to nerve growth factor in the WEHI and Ordway lines (NGF: 50ng/ml, 2.1- and 13.4-fold increases, respectively, P< or =0.05). mRNA for NR2A or NR2B was not detected in any PC12 cell-line. NR2C mRNA expression varied between lines and increased after NGF treatment (approximately 4-fold increase in WEHI and Ordway lines, P< or =0.05). In the Ordway line, NR2D mRNA was seen only after NGF treatment. Immunohistochemistry confirmed protein expression for NR1, NR2C and NR2D, and while fluorescence intensity changes in response to NGF paralleled mRNA responses, the degree of increase was of reduced magnitude. Whole-cell patch-clamping of NGF treated cells failed to detect functional NMDA receptors in any of the cell-lines. Our study demonstrates that in contrast to neurons from the CNS, PC12 cells do not express a normal complement of NMDA receptor-subunits, and this may be one factor limiting functional responses to NMDA/glutamate and consequently the use of PC12 cells as a neuronal model.     

Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure

Long-term deficits in cognitive function are the principal effects of lead (Pb2+) exposure in children and can be modeled in experimental animals. Current therapeutic approaches in the treatment of childhood Pb2+ intoxication are not effective in reversing learning deficits once they have occurred. We report that environmental enrichment reverses long-term deficits in spatial learning produced by developmental Pb2+ exposure in rats. Enhanced learning performance of Pb2+-exposed animals reared in an enriched environment was associated with recovery of deficits in N-methyl-D-aspartate receptor subunit 1 (NR1) mRNA and induction of brain-derived neurotrophic factor (BDNF) mRNA in the hippocampus. The effect of environmental enrichment on NR1 and BDNF gene expression was specific to Pb2+-exposed animals and was present in the absence of changes in the NR2B subunit of the N-methyl-D-aspartate receptor, GluR1, alpha CamKII, or PSD-95 gene expression measured in the same animals. Our findings demonstrate that the learning impairments and NR1 subunit mRNA deficits resulting from developmental Pb2+ exposure are reversible if the animals are provided with an enriched environment even after the exposure has occurred. We propose environmental enrichment as a basis for the treatment of childhood Pb2+ intoxication.     

Lead exposure during synaptogenesis alters NMDA receptor targeting via NMDA receptor inhibition

N-methyl-D-aspartate receptor (NMDAR) ontogeny and subunit expression are altered during developmental lead (Pb2+) exposure. However, it is unknown whether these changes occur at the synaptic or cellular level. Synaptic and extra-synaptic NMDARs have distinct cellular roles, thus, the effects of Pb2+ on NMDAR synaptic targeting may affect neuronal function. In this communication, we show that Pb2+ exposure during synaptogenesis in hippocampal neurons altered synaptic NMDAR composition, resulting in a decrease in NR2A-containing NMDARs at established synapses. Conversely, we observed increased targeting of the obligatory NR1 subunit of the NMDAR to the postsynaptic density (PSD) based on the increased colocalization with the postsynaptic protein PSD-95. This finding together with increased binding of the NR2B-subunit specific ligand [3H]-ifenprodil, suggests increased targeting of NR2B-NMDARs to dendritic spines as a result of Pb2+ exposure. During brain development, there is a shift of NR2B- to NR2A-containing NMDARs. Our findings suggest that Pb2+ exposure impairs or delays this developmental switch at the level of the synapse. Finally, we show that alter expression of NMDAR complexes in the dendritic spine is most likely due to NMDAR inhibition, as exposure to the NMDAR antagonist aminophosphonovaleric acid (APV) had similar effects as Pb2+ exposure. These data suggest that NMDAR inhibition by Pb2+ during synaptogensis alters NMDAR synapse development, which may have lasting consequences on downstream signaling.     

Accurate quantification of the mRNA of NMDAR1 splice variants measured by competitive RT-PCR

N-methyl-D-aspartate receptors (NMDAR) belong to the subclass of ionotropic glutamate receptors and are widely distributed in the vertebrate brain. Molecular cloning has revealed the existence of six NMDAR subunits: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D) and one NMDAR3A (NR3A). Alternative splicing of the single NR1 gene generates eight isoforms with distinct functional properties [M. Hollmann, J. Boulter, C. Maron, L. Beasley, J. Sullivan, G. Pecht, S. Heinemann, Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor, Neuron 10 (1993) 943-954 [8]; R.S. Zukin, M.V.L. Bennett, Alternatively spliced isoforms of the NMDAR1 receptor subunit, TiNS 18 (1995) 306-313 [20]]. Despite the progress made in the functional analysis of NMDARs the molecular architecture of this receptor remains to be elucidated. In situ hybridization studies have already indicated that splicing of the NR1 gene is regionally regulated in the rodent brain, which may contribute to functional diversity of NMDARs in distinct brain areas [D.J. Laurie, P.H. Seeburg, Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA, J. Neurosci. 14 (1994) 3180-3194 [10]; D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney Jr., Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurology 343 (1994) 1-16 [18]; M. Hollmann, S. Heinemann, Cloned glutamate receptors, Ann. Rev. Neurosci. 17 (1994) 31-108 [9]]. Since in situ hybridization techniques do not allow accurate quantification of distinct NR1 splice variants and are also very time-consuming, an accurate and sensitive competitive RT-PCR assay was developed. This method was then used to study the distribution of three NR1 splice variants in the rat brain, and the results are compared with former in situ hybridization studies.     

Cellular and Subcellular Distribution of NMDAR1 Splice Variant mRNA in the Rat Lumbar Spinal Cord

The regional distribution of alternatively spliced messenger RNA encoding the N -methyl-D-aspartate (NMDA) receptor R1 subunit (NMDAR1) variants was examined by in situ hybridization in the rat lumbar spinal cord. Splice-specific oligonucleotide probes [recognizing full-length mRNA (NMDAR1-1), deletion exon 21 (NMDAR1-2), deletion exon 22 (NMDAR1-3), combined deletion exons 21 and 22 (NMDAR1-4) and mRNA which lacks (NMDAR1-a) or contains exon 5 (NMDAR1-b)] detected marked differences in abundance and distribution of N- and C-terminal spliced variants. The NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs were evenly distributed throughout all laminae of the dorsal and ventral horns. In the superficial dorsal horn NMDAR1-b mRNA was preferentially detected in laminae II inner and III, while NMDAR1-1 mRNA was restricted to laminae I to III. Large neurons in laminae IV and V contained mainly NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs and occasionally NMDAR1-b. The NMDAR1-3 variant was only detected in very low abundance, being restricted to occasional cells in lamina I and II. In the ventral horn, motor neurons showed strong signals for NMDAR1-a, NMDAR1-b, NMDAR1-2 and NMDAR1-4 mRNAs. Serial sectioning through large motor neurons permitted the detection of multiple splice variants in single neurons. Analysis of the subcellular distribution of the mRNAs revealed that the NMDAR1-1 mRNA was almost exclusively found in the cell nucleus, NMDAR1-a mRNA was largely in the cytoplasm, while all other splice variants showed a homogeneous distribution between nucleus and cytoplasm. Comparison of the in situ hybridization images with functional analyses of heteromeric recombinant receptors will be necessary to ascertain whether splice variants of the NMDAR1 receptor subunit can account for some of the known electrophysiological properties of spinal cord neurons under physiological and pathophysiological conditions.     

Unique levels of expression of N-methyl-D-aspartate receptor subunits and neuronal nitric oxide synthase in the rostral ventrolateral medulla of the spontaneously hypertensive rat

The rostral ventrolateral medulla (RVLM) is the major brainstem region contributing to sympathetic control of blood pressure. We have compared the expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A-D), NR1 splice variants (NR1-1a/1b, -2a/2b, -3a/3b, -4a/4b), and the neuronal and inducible isoforms of NO synthase (nNOS and iNOS) in the RVLM of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR), based on the hypothesis that altered NMDA receptor make-up or altered expression of endogenous NO may be associated with the increase in sympathetic output described from this site in hypertension. Total RNA was extracted and reverse transcribed from the RVLM of mature male WKY and SHR (16-23 weeks). Conventional polymerase chain reaction (PCR) indicated that only the NR1 splice variants NR1-2a, NR1-2b, NR1-4a and NR1-4b were expressed in the RVLM of either species. Quantitative real-time PCR indicated that for both strains of rat, mRNA for the NR1 subunit (all splice variants) was the most abundant (16.5-fold greater, P< or =0.05, relative to the NR2A subunit). Amongst the NR2A-D subunits, NR2C was the most abundant (7- and 1.7-fold greater relative to the NR2A subunit, P< or =0.05, WKY and SHR, respectively). Relative to WKY, mRNA levels for the NR2C and NR2D subunits in the SHR RVLM were significantly lower (0.3- and 0.25-fold less, P< or =0.05), while nNOS was significantly higher (1.76-fold greater, P< or =0.05). This was confirmed immunohistochemically for nNOS expression. These results demonstrate differential expression levels of NMDA receptor subunits and NOS isoforms in the RVLM region of SHR when compared to WKY rats.     

Chronic ethanol exposure and withdrawal influence NMDA receptor subunit and splice variant mRNA expression in the rat cerebral cortex

Chronic ethanol exposure and subsequent withdrawal are known to change NMDA receptor activity. This study examined the effects of chronic ethanol administration and withdrawal on the expression of several NMDA receptor subunit and splice variant mRNAs in the rat cerebral cortex. Ethanol dependence was induced by ethanol vapour exposure. To delineate between seizure-induced changes in expression during withdrawal and those due to withdrawal per se, another group of naive rats was treated with pentylenetetrazol (PTZ) injection (30 mg/kg, i.p.). RNA samples from the cortices of chronically treated and withdrawing animals were compared to those from pair-fed controls. Changes in NMDA receptor mRNA expression were determined using ribonuclease protection assays targetting the NR2A, -2B, -2C and NR1-pan subunits as well as the three alternatively spliced NR1 inserts (NR1-pan describes all the known NR1 splice variants generated from the 5' insert and the two 3' inserts). The ratio of NR1 mRNA incorporating the 5' insert vs. that lacking it was decreased during ethanol exposure and up to 48 h after withdrawal. NR2B mRNA expression was elevated during exposure, but returned to control levels 18 h after withdrawal. Levels of NR2A, NR2C, NR1-pan and both 3' NR1 insert mRNAs from the ethanol-treated groups did not alter compared with the pair-fed control group. No changes in the level of any NMDA receptor subunit mRNA was detected in the PTZ-treated animals. These data support the hypothesis that changes in NMDA receptor subunit composition may underlie a neuronal adaptation to the chronic ethanol-inhibition and may therefore be important in the precipitation of withdrawal hyperactivity.     

Differential expression of alternatively spliced isoforms of neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptors (NMDAR) in knockout mice deficient in nNOS alpha (nNOS alpha(Delta/Delta) mice)

Recent data suggest that the neuronal isoform of nitric oxide synthase (nNOS) and glutamate receptors of the N-methyl-D-aspartate (NMDA) type are physically coupled and, hence, functionally interrelated. Several alternatively spliced isoforms of the N-methyl-D-aspartate receptor 1 (NMDAR1) subunit and the neuronal nitric oxide synthase (nNOS) are known, and recent studies have shown that a spliced C-terminal may be responsible for the coupling of NMDAR's to nNOS via its PDZ domain and the postsynaptic density protein PSD95. However, little is known about whether and to what extent changes in nNOS expression influence NMDA receptor density or function. We have therefore compared the localization of nNOS alpha, beta and gamma with that of two relevant NMDAR1 splice variants in wild-type mice versus knockout mice deficient in nNOS alpha, generated by homologous recombination with a targeted deletion of exon 2, containing one PDZ domain (nNOS alpha(Delta/Delta) mice). Whereas nNOS alpha was completely absent in nNOS alpha(Delta/Delta) mice, nNOS beta and gamma were expressed in both wild-type and knockout animals. nNOS gamma mRNA, though, was hardly detectable, if at all, mainly within the olfactory bulb, the cerebellum and mesencephalic nuclei of knockout animals. The expression of the NMDAR1-1 splice variant (without any short carboxy-terminal amino acid motif, recognized by PDZ domains) was remarkably decreased in striatal, cortical, hippocampal and cerebellar tissue in nNOS alpha(Delta/Delta) animals, but no changes in NMDAR1-4 (with an alternatively spliced C-terminal and thus with a PDZ binding motif) mRNA and protein levels were observed. While NMDAR1-4 may be related to receptor targeting and clustering to PSD95 and to nNOS, our data suggest that differences in nNOS expression obviously do not directly influence gene expression of this particular NMDAR splice variant. Otherwise, the observed diminution of NMDAR1-1 splice variant mRNA and protein levels may, at least partially, explain the decreased vulnerability of nNOS alpha(Delta/Delta) mice to glutamate-mediated neurotoxicity.     

Estradiol potentiation of NR2B‐dependent EPSCs is not due to changes in NR2B protein expression or phosphorylation

The hormone, 17β‐estradiol (E2), influences the structure and function of synapses in the CA1 region of the hippocampus. E2 increases the density of dendritic spines and excitatory synapses on CA1 pyramidal cells, increases CA1 cells' sensitivity to excitatory synaptic input mediated by the NMDA receptor (NMDAR), enhances NMDAR‐dependent long‐term potentiation, and improves hippocampus‐dependent working memory. Smith and McMahon ( 2006 J Neurosci 26:8517–8522) reported that the larger NMDAR‐mediated excitatory postsynaptic currents (EPSCs) recorded after E2 treatment are due primarily to an increased contribution of NR2B‐containing NMDARs. We used a combination of electrophysiology, Western blot, and immunofluorescence to investigate two potential mechanisms by which E2 could enhance NR2B‐dependent EPSCs: An increase in NMDAR subunit protein levels and/or a change(s) in NR2B phosphorylation. Our studies confirmed the E2‐induced increase in NR2B‐dependent EPSC amplitude, but we found no evidence that E2 affects protein levels for the NR1, NR2A, or NR2B subunit of the NMDAR, nor that E2 affects phosphorylation of NR2B. Our findings suggest that the effects of E2 on NMDAR‐dependent synaptic physiology in the hippocampus likely result from recruitment of NR2B‐containing NMDARs to synapses rather than from increased expression of NMDARs or changes in their phosphorylation state. © 2010 Wiley‐Liss, Inc.     

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state. We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase. Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites. Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.     

Developmental expression of N-methyl-D-aspartate glutamate receptor 1 splice variants in the chick retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. The N-methyl-D-aspartate glutamate receptor (NMDAR) is assembled as a tetramer containing NR1 and NR2, and possibly NR3 subunits, NR1 being essential for the formation of the ion channel. The NMDAR1 (NR1) gene encodes for mRNAs that generate at least eight functional variants by alternative splicing of exon 5 (cassette N1), 21 (cassette C1), or 22 (cassettes C2 or C2'). NR1 splice variants were identified in the mature chick retina, and their variation during embryonic development (ED) was analyzed. NR1 was shown to lack N1 in early ED, shifting to N1-containing variants in the mature retina, which could contribute to explaining the distinct biochemical properties of retinal NMDARs compared with the CNS. Sequence analysis of C-terminal variants containing C1 and C2 cassettes suggests a membrane-targeting mechanism for avian NMDARs distinct from that in mammals. An NR1 variant containing a novel alternative C-terminal splice exon named C3 was found, which encodes six amino acids containing a predicted casein kinase II phosphorylation site. This new variant is expressed in the retina during a restricted period of ED, coincident with the generation of spontaneous calcium activity waves, which precedes synapse formation in the retina, suggesting its participation in this process.     

The effects of aging on different C-terminal splice forms of the zeta1(NR1) subunit of the N-methyl-d-aspartate receptor in mice

Changes in NMDA receptors in the prefrontal/frontal cortex and hippocampus of C57BL/6 mice during aging show a relationship to declines in spatial learning. The present study was designed to determine whether aging influences the mRNA expression of different splice forms of the zeta1 subunit of the NMDA receptor. We examined the mRNA of 4 C-terminal splice forms with the use of in situ hybridization. The zeta1-1 splice form (+C1 and +C2 cassettes) overall showed a maintenance of mRNA density from 3 to 10 months of age, followed by a significant decline by 26 months of age. In contrast, the mRNA for the zeta1-3 splice form [+C1 and +C2'(-C2)] showed significant declines between 3- and 10-month-old mice. These declines were maintained in the old mice. The zeta1-2 splice form (-C1 and +C2) showed a near-significant decrease in expression during aging across all brain regions. The zeta1-4 subunit mRNA [-C1 and +C2' (-C2)] showed no significant changes with increased age. These results indicate that there is a differential effect of aging on different splice variants of the zeta1 subunit of the NMDA receptor and those that are affected show a different temporal pattern of aging. This heterogeneity has implications for producing imbalances in the modulation of the remaining receptors.     

N-methyl-D-aspartate receptor mRNA levels change during reproductive senescence in the hippocampus of female rats.

Estrogen interacts with N-methyl-d-aspartate (NMDA) receptors to regulate multiple aspects of morphological and functional plasticity. In the hippocampus, estrogens increase both dendritic spine density and synapse number, and NMDA antagonists block these effects. This plasticity in the hippocampus mediated by estrogen may be of particular importance in the context of aging when estrogen levels change and cognitive function is often impaired. Therefore, the present study was designed to investigate effects of aging and reproductive status on NMDA receptor (NR) subunit mRNA levels in the hippocampus. NR1, NR2A, and NR2B mRNA levels were measured by RNase protection assay in young (3-4 month), middle-aged (12-13 month), and aged (24-25 month) Sprague-Dawley rats in different phases of the estrous cycle in cycling animals and in acyclic subjects. Our results demonstrated that NMDA receptor subunit mRNA levels were much more prominently affected by the chronological age than by the reproductive status of the animals. Age-related changes were observed in NR1, NR2A, and NR2B in the ventral hippocampus and in NR1 and NR2B in the dorsal hippocampus. However, the only relationship with reproductive status was seen for NR1 mRNA, and this was restricted to the ventral hippocampus. An interaction between chronological age and reproductive status was found, with higher levels of NR1 mRNA seen in young animals in proestrus than in those in diestrus I (high and low estrogen levels, respectively). However, this relationship was not seen in the aged subjects. These results demonstrate that the hippocampus is subjected to age-related alterations in NMDA receptor subunit mRNA levels and that animals of different ages are influenced differently by reproductive status. This shift in the NMDA receptor mRNA levels may be a possible molecular mechanism contributing to alterations in cognitive behavior during normal aging.