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.     

Selective decrease in NR1 subunit splice variant mRNA in the hippocampus of Pb2+-exposed rats: implications for synaptic targeting and cell surface expression of NMDAR complexes

We have previously shown that exposure to environmentally relevant levels of Pb(2+) during brain development decreases the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) and NR2A genes in the hippocampus of young adult rats and was associated with deficits in hippocampal LTP and spatial learning [Neuroscience 99 (2000) 233-242]. In the present study, we demonstrate that the lower levels of NR1 subunit mRNA expressed in the Pb(2+)-exposed hippocampus are principally due to decreased levels of the NR1-4 and NR1-2 splice variants. These changes were present in the absence of changes in GluR1, PSD-95 and alphaCaMKII gene expression. A unique characteristic of these splice variants is that they lack the C1 cassette. Further, these splice variants have been shown to impart the highest cell surface expression, PKC potentiation and calcium kinetics to NMDAR complexes. Our present findings indicate that Pb(2+)-induced changes in NR1 subunit splice variant mRNA expression in the hippocampus may provide a mechanism by which Pb(2+)-exposure can modify NMDAR-mediated calcium signaling and influence the degree of synaptic plasticity.     

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.     

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.     

Lead exposure alters cyclic-AMP response element binding protein phosphorylation and binding activity in the developing rat brain

We examined the effect of lead (Pb(2+)) exposure during development on cyclic-AMP response element binding protein (CREB) expression and phosphorylation in cortical and hippocampal nuclear extracts at postnatal (PN) days 7, 14, 21 and 50. We also examined the binding of CREB family proteins to the cyclic-AMP response element (CRE) using a novel filter-binding assay that provides a quantitative measure of binding kinetics. In the hippocampus and cerebral cortex of control rats, CREB and phospho-CREB (pCREB; serine-133) expression is highest at PN7 and decreases steadily until PN50. Developmental Pb(2+) exposure does not affect total CREB levels but decreases pCREB levels at PN14 and PN50 in the cortex and at PN50 in the hippocampus. Using the filter-binding assay, we measured a 30% decrease in B(max) and 38% decrease in the Kd of CREB family proteins for the CRE in PN50 hippocampal nuclear fractions prepared from Pb(2+)-exposed rats. A similar, but nonsignificant, trend is observed in the cortex of PN50 lead-exposed rats. In addition, a 70% increase in the B(max) was observed in the cortex of PN14 lead-exposed rats without a significant change in the Kd. These disruptions in pCREB expression and binding activity of CREB family members during the ontogeny of the rat brain begin to decipher intracellular mechanisms of Pb(2+) neurotoxicity.     

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.     

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.     

Calcium/calmodulin-dependent protein kinase II activity and expression are altered in the hippocampus of Pb2+-exposed rats

In the present study, we examined whether calcium/calmodulin-dependent protein kinase II (CaMKII) is affected by chronic developmental Pb2+ exposure. The effects of Pb2+ exposure on rat hippocampal CaMKII were assessed by measuring CaMKII activity, phosphorylation of CaMKII at threonine-286, and CaMKII alpha and beta protein levels. In the hippocampus of Pb2+-exposed 50-day-old rats known to exhibit deficits in hippocampal long-term potentiation (LTP) and spatial learning, there was a marked reduction (41%) in the apparent maximal velocity (Vmax) of CaMKII and a significant increase (22%) in apparent affinity of the enzyme. These Pb2+-induced changes in CaMKII activity could not be explained by changes in enzyme phosphorylation at threonine-286 or sensitivity to calmodulin. In vitro incubation of hippocampal homogenates from control rats, but not from Pb2+-exposed rats, with Pb2+ prior to assay recapitulated the increase in the affinity of the enzyme observed with in vivo exposure to Pb2+. Western blots of cytosolic and membrane fractions from hippocampus showed a significant decrease in the levels of CaMKII-beta but not alpha protein in the cytosolic fraction of Pb2+-exposed rats. These findings indicate effects of developmental Pb2+ exposure on CaMKII, a component of calcium signaling associated with synaptic plasticity, learning, and memory.     

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.     

Expression of N-Methyl-D-Aspartate receptor subunit mRNAs in the human brain: Hippocampus and cortex

N-methyl-D-aspartate receptor (NR) activation in the hippocampus and neocortex plays a central role in memory and cognitive function. We analyzed the cellular expression of the five NR subunit (NR1 and NR2A-D) mRNAs in these regions with in situ hybridization and human ribonucleotide probes. Film autoradiograms demonstrated a distinct pattern of hybridization signal in the hippocampal complex and the neocortex with probes for NR1, NR2A, and NR2B mRNA. NR2C and NR2D probes yielded scattered signals without a distinct organization. At the emulsion level, the NR1 probe produced high-density hybridization signals across the hippocampal complex. NR2A mRNA was higher in dentate granule cells and pyramidal cells in CA1 and subiculum compared to hilus neurons. NR2B mRNA expression was moderate throughout, with higher expression in dentate granule cells, CA1 and CA3 pyramidal cells than in hilus neurons. In the hippocampal complex, the NR2C probe signal was not different from background in any region, whereas the NR2D probe signal resulted in low to moderate grain densities. We analyzed NR subunit mRNA expression in the prefrontal, parietal, primary visual, and motor cortices. All areas displayed strong NR1 hybridization signals. NR2A and NR2B mRNAs were expressed in cortical areas and layers. NR2C mRNA was expressed at low levels in distinct layers that differed by region and the NR2D signal was equally moderate throughout all regions. Pyramidal cells in both hippocampus and neocortex express NR1, NR2A, NR2B, and, to a lesser extent, NR2D mRNA. Interneurons or granular layer neurons and some glial cells express NR2C mRNA. J. Comp. Neurol. 390:75–90, 1998. © 1998 Wiley-Liss, Inc.     

Single cell RT-PCR demonstrates differential expression of GABAC receptor rho subunits in rat hippocampal pyramidal and granule cells

Although gamma-aminobutyric acid (GABA)C receptor rho1, rho2 and rho3 subunits are reportedly expressed in pyramidal and granule cells in the hippocampus at various developmental stages, it is not clear whether these three rho subunits are coexpressed in a single neuron. To attempt to answer this question, we performed single-cell RT-PCR for rho subunits from neurons of rat brain hippocampus. In hippocampal cultures, pyramidal cells were positive for rho1 mRNA expression in 89%, rho2 in 94% and rho3 in 94%, while granule cells were positive for rho1 mRNA in only 6%, rho2 in 36% and rho3 in 91%. Intensive amplification of the RT-PCR products by the second PCR revealed that all the three rho subunits were coexpressed in a single pyramidal and granule cells from both of the cultures and the slices. These results suggest that all the three GABAC receptor rho1, rho2 and rho3 subunits are present probably in different compositions in pyramidal and granule cells in the rat hippocampus.     

Hippocampal N-methyl-D-aspartate receptor subunit mRNA levels in temporal lobe epilepsy patients.

Changes in the subunit stoichiometry of the N-methyl-D-aspartate (NMDA) receptor (NMDAR) alters its channel properties, and may enhance or reduce neuronal excitability in temporal lobe epilepsy patients. This study determined whether hippocampal NMDA receptor subunit mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsy cases. Hippocampal sclerosis (HS; n = 16), non-HS (n = 10), and autopsy hippocampi (n = 9) were studied for NMDAR1 (NR1) and NR2A-D mRNA levels by using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, non-HS and HS patients showed increased NR2A and NR2B hybridization densities per dentate granule cell. Furthermore, non-HS hippocampi showed increased NR1 and NR2B mRNA levels per CA2/3 pyramidal neuron compared with autopsy cases. HS patients, by contrast, showed decreased NR2A hybridization densities per CA2/3 pyramidal neuron compared with non-HS and autopsy cases. These findings indicate that chronic temporal lobe seizures are associated with differential changes in hippocampal NR1 and NR2A-D hybridization densities that vary by subfield and clinical-pathological category. In temporal lobe epilepsy patients, these findings support the hypothesis that in dentate granule cells NMDA receptors are increased, and excitatory postsynaptic potentials should be strongly NMDA mediated compared with nonseizure autopsies. HS patients, by comparison, showed decreased pyramidal neuron NR2A mRNA levels, and this suggests that NMDA-mediated pyramidal neuron responses should be reduced in HS patients compared with non-HS cases.     

咖啡因对慢性低O2高CO2大鼠学习记忆及N-甲基-D-天冬氨酸受体亚基1 mRNA表达的影响

目的：探讨咖啡因对慢性低O2高CO2处理的大鼠空间学习记忆、皮质和海马N-甲基-D-天冬氨酸受体（NMDAR）亚基1mRNA（NR1 mRNA）表达的影响。方法：SD大鼠48只分为4组,正常对照组;低O2高CO24周（HH）组,处理组：低O2高CO2＋咖啡因0．1mg·mL^-14周（HCl组）,低O2高CO2＋咖啡因0．3mg·mL^-14周（HC2组）。处理组以咖啡因水溶液干预4周后行Morris水迷宫实验,观察大鼠寻找站台的平均逃避潜伏期和游泳总距离;采用原位杂交法观察大鼠海马及皮质区NRImRNA的表达与分布情况。结果：①Morris水迷宫实验显示,HH组与对照组相比大鼠寻找站台的平均逃避潜伏期延长、游泳总距离增加（P〈0．05）,HC2组有显著性降低（P〈0．05）;②原位杂交显示NR1 mRNA阳性细胞广泛分布于海马和皮质区;模型组与对照组比较大鼠海马锥体细胞层NR1 mRNA表达的平均吸光度值降低（P〈0．05）,而咖啡因处理组平均吸光度值升高。结论：咖啡因可改善慢性低O2高CO2大鼠的空间学习记忆能力并增加海马和皮质区NR1 mRNA的表达。     

Lead inhibition of NMDA channels in native and recombinant receptors

NMDA channels are key targets for lead (Pb2+) neurotoxicity and Pb2+-induced inhibition of NMDA current is age- and subunit-dependent. In rat cerebellar granule cells maintained in high KCl, glycine affinity as well as sensitivity to ifenprodil change significantly with the days in vitro, indicating a reduction of NR2B subunit expression. Pb2+ blocked NMDA current with IC50 approximately 4 microM and this effect decreased significantly during the second week in vitro. In Xenopus laevis oocytes expressing recombinant NR1-NR2A, NR1-NR2B or NR1-NR2C receptors, Pb2+ inhibited glutamate-activated currents with IC50 of 3.3, 2.5 and 4.7 microM respectively. These data indicate that Pb2+ action is dependent on subunit composition and suggest that down-regulation of the NR2B subunit is correlated to a diminished sensitivity to Pb2+ inhibition.     

局灶性脑缺血后海马各区NMDAR表达的实验研究

目的：观察局灶性脑缺血对N－甲基－D－天门冬氨酸受体（NMDAR）及NMDARmRNA表达的影响。方法：采用线栓法制作可复流脑中动脉闭塞大鼠模型。30只雄性大鼠随机分为3组;正常组、假手术组和大脑中动脉闭塞（MCAO）组。MCADO后第5天处死动物,取脑组织进行NR1免疫组化及NR1mRNA原们杂交检测。结果（1）正常组海马各区均有NR1阳性细胞伯分布,其中海马CA3区、DG区分布较多;MCAO组NR1阳性细胞在海马CA1区、CA3区及DG区的数密度、光密度较正常组高;（2）正常组海马各区均有极少量的NR1mRNA阳性细胞分布,MCAO组NR1mRNA阳性细胞在海马CA1区、CA3区的数密度、光密度均较正常组有显著性增高。结论：大鼠海马各区都存在着NR1及NR1mRNA不同程度的表达,MCAO后NR1及NR1mRNA的表达水平上调。     

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.