Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.     

Transient expression of NMDA receptor subunit NR2B in the developing rat heart

NMDA receptors represent a subtype of the ionotropic glutamate receptor family, comprising three classes of subunits (NR1, NR2A-D, NR3), which exhibit distinct patterns of regional and developmental expression in the CNS. Recently, some NMDA receptor subunits have also been described in adult extraneuronal tissues and keratinocytes. However, their developmental expression patterns are currently unknown. With use of RT-PCR and western blot analysis, the expression of NMDA receptor subunit NR2B was investigated in the developing rat heart. NR2B mRNA and protein were detected in heart tissue of rats from embryonic day 14 until postnatal day 21 but disappeared 10 weeks after birth. In contrast, no NMDA receptor subunit NR1, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR2, or anchoring postsynaptic density protein-95 could be detected in rat heart at any developmental stage. Confocal microscopy of cultured cardiac myocytes (CMs) from neonatal rats revealed distinct NR2B staining mainly of intracellular structures. However, no functional NMDA receptor could be detected on CMs by whole-cell recordings. In conclusion, high concentrations of NR2B protein can be detected in early rat heart development, but its function still remains elusive.     

Immunolocalization and Biochemical Characterization of N-methyl-D-aspartate Receptor Subunit NR1 from Rat Brain

The N-methyl-D-aspartate (NMDA) receptor subunit NR1 gene can produce eight isoforms in rat brain. A novel methodology for purifying NMDA receptor NR1 subunit from rat brain is reported here using chicken polyclonal antibodies (IgYs) against synthetic peptides corresponding to N1, C1 and C2′ cassettes. The isolated protein was recognized by produced IgYs and commercial anti-NR1 IgGs, shown by MALDI-TOF MS a MW = 131,192 Da (glycosylated form); the enzymatically deglycosylated protein revealed a MW = 102,754 Da. The NMDA receptor NR1 subunit was characterized as being a heavily N-glycosylated protein. The isoelectric point was determined (6.3) as being different from that predicted for any of the isoforms (7.9–9.02). Attempts to separate the isoforms from the purified NR1 were unsuccessful, indicating the presence of just one isoform (NR1₁₁₁). Immunohistochemistry on hippocampus regions CA1, CA3 and Dentate gyrus with anti-N1, anti-N2 and anti-C2′ IgYs showed different staining intensity, depending upon the antibody assayed.     

Orexin decreases mRNA expressions of NMDA and AMPA receptor subunits in rat primary neuron cultures

Orexin is one of the orexigenic neuropeptides in the hypothalamus. Orexin neurons in the lateral hypothalamus (LH) project into the cerebral cortex and hippocampus in which the receptors are distributed in high concentrations. Therefore, to elucidate the actions of orexin in the cerebral cortex, we examined its effects on the mRNA expressions of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A, NR2B) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits (GluR1, GluR2) following 6-day application of orexin-A or orexin-B to rat primary cortical neuron cultures. The mRNAs of NR1 and NR2A subunits were significantly decreased by orexin-A and orexin-B at concentrations over 0.1 μM and 0.01 μM, respectively. The mRNA expression of NR2B subunit was also significantly decreased by orexin-A and orexin-B only at the concentration of 1 μM. Moreover, orexin-A and orexin-B at concentrations over 0.01 μM significantly decreased the mRNA expressions of AMPA receptor subunits, GluR1 and GluR2. The present study demonstrated that orexins significantly suppressed RNA expressions of NMDA and AMPA receptor subunits in cortical neuron cultures, suggesting that orexin may regulate the higher functions of the cerebral cortex as well as be involved in energy regulation in the hypothalamus.     

Characterization in cultured cerebellar granule cells and in the developing rat brain of mRNA variants for the NMDA receptor 2C subunit

N-Methyl-D-aspartate (NMDA) receptors are heteromeric structures resulting from the association of at least two distantly related subunit types, NR1 and one of the four NR2 subunits (NR2A-NR2D). When associated with NR1, the NR2 subunits impose specific properties to the reconstituted NMDA receptors. Although the NR1 mRNAs are expressed in the majority of central neurons, the NR2 subunits display distinct patterns of expression in the developing and adult rat brain. The NR2C subunit is barely expressed in the rat forebrain, whereas its expression increases substantially in the granule cells in the course of cerebellar development. We have identified novel NR2C splice variants in cultured cerebellar granule cells as well as in the developing cerebellum. When compared with the prototypic NR2C mRNA, these variants carry one (NR2Cb) or two (NR2Cd) insertions or a deletion (NR2Cc) and encode putative NR2C polypeptides that terminate between the third and fourth membrane segments or between the first and second membrane segments. RT-PCR analysis and in situ hybridization show that expression of the splice variants is developmentally regulated, both in the cerebellum and in the hippocampus. Electrophysiological recordings and microfluorimetry emissions in transfected human embryonic kidney 293 cells indicate that the NR2Cb variant, when expressed in combination with NR1, does not contribute to the formation of functional receptor channels. The significance of theses findings is discussed.     

The effect of postnatal manganese exposure on the NMDA receptor signaling pathway in rat hippocampus

Overexposure to manganese (Mn) is associated with neurological disorders in children. Evidence indicated that N‐methyl‐d ‐aspartate (NMDA) receptor signaling pathway was critical for neurobehavioral function. However, whether NMDA receptor signaling pathway contributes to Mn‐induced neurotoxicity remains unknown. In this study, newborn Sprague–Dawley rats were randomly assigned to four groups exposed to 0, 10, 20, and 30 mg/kg of Mn²⁺ by intraperitoneal injection (n = 10/group: five males and five females). After 3 weeks of Mn exposure, messenger RNA (mRNA) and protein expression of NMDA receptor subunits (NR1, NR2A, and NR2B), cAMP‐response element binding protein (CREB), and brain‐derived neurotrophic factor (BDNF) in hippocampus were measured by real‐time quantitative RT‐PCR and Western blot. In Mn‐exposed rats, decreased mRNA and protein expression of NR1, NR2A, and NR2B, CREB, and BDNF was observed. The results imply that downregulated NMDA receptor signaling pathway may be of vital importance in the neuropathological process of Mn‐induced neurotoxicity.     

Age and Visual Experience-dependent Expression of NMDAR1 Splice Variants in Rat Retina

The N-methyl-D-aspartate receptor (NMDAR) is a key molecule mediating brain plasticity related processes. Knowing that alternative splicing of the NMDAR1 (NR1) subunit offers molecular diversity to NMDAR, controls the forward trafficking of the NR1 protein and is important for placing NMDA receptors at synapses, we investigated herein the postnatal developmental expression and the influence of visual deprivation on NR1 subunit splice variants in rat retina. Real-time PCR was performed using oligonucleotide primers specific for N- terminal (NR1a, NR1b) and C-terminal splice variants (NR1-1, NR1-2, NR1-3, NR1-4). The developmental profiles of mRNA expression levels of all NR1 isoforms peaked at the end of the third week. Dark rearing led to reductions in both N- and C-terminal NR1 variants in several developmental ages and a significant interaction between age and visual experience was observed for NR1a, NR1-2 and NR1-4 expression. Our results have demonstrated a developmental and visual experience-dependent regulation of NR1 splicing in rat retina.     

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.     

Developmental and Regional Expression of NMDA Receptor Subtypes Containing the NR2D Subunit in Rat Brain

Abstract: The regional and developmental expression of NMDA receptors containing the NR2D subunit was analyzed on the level of the subunit mRNA and protein in rat brain. RNase protection experiments indicated that among two proposed splice variants of the NR2D subunit, only the NR2D-2 subunit is expressed. The regional distribution of the NR2D subunit protein was visualized with a newly developed NR2D-2 subunit-specific antiserum on brain sections using the histoblot technique. In adult brain, NR2D immunoreactivity was mainly restricted to diencephalic, mesencephalic, and brainstem structures. During postnatal development, the NR2D subunit was detected transiently in certain regions, such as the ventro-basal complex of the thalamus, hippocampus, inferior colliculus, and brainstem reticular formation, suggesting that NR2D subunit-containing receptors play a role in these brain areas only during development. The level of NR2D subunit mRNA and protein decreased during late postnatal development. However, significant levels of NR2D subunit mRNA and protein were present in adulthood, in particular, in the globus pallidus, thalamus, subthalamic nuclei, and superior colliculus. These results indicate a functional relevance for NMDA receptors containing the NR2D subunit in the developing and adult brain, although its expression in the adult brain is less prominent and restricted to a few brain areas.     

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.     

癫痫大鼠海马NMDA受体亚基NR2A和BDNFmRNA水平的检测

目的通过锂一匹罗卡品癫痫模型（ithium—pilocarpine seizures rats model of epilepsy,LPS）,研究NMDA受体亚基NR2A、BDNF mRNA的表达,探讨NR2A、BDNF在LPS中的作用。方法建立氯化锂-匹罗卡品大鼠模型,运用原位杂交技术检测致痫后各组不同时间点海马CAI、CA3及DG区NR2A与BDNF mRNA的表达。结果LPS海马NR2A、BDNF mRNA在各观察时间点及部位模型组与正常对照组比较均有明显上调,且有显著统计学差异（P〈0．05）。模型组NR2A mRNA的表达上调7d达峰值（P〈0．05）;而BDNF mRNA表达上调14d达峰值。VPA干预组NR2A mRNA在大鼠海马不同时间及部位（除1d的CA3区）的表达较模型组明显下调（P〈0．05）;BDNF mRNA在大鼠海马不同时间及部位（除28d的DG区）的表达较模型组明显下调（P〈0．05）。结论锂－匹罗卡品腹腔注射可诱导大鼠海马NR2A和BDNF mRNA的表达明显上调;NR2A mRNA表达的增强可能是诱导调控BDNF mRNA表达增强的重要机制之一,说明NMDA受体亚基NR2A可能成为抑制癫痫发作的新靶点。     

Spreading depression-induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in alpha-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12-15-fold increase in alpha7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the alpha4 nAChR subunit was not altered. These data point to the alpha7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating alpha7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).     

Chronic Unpredictable Stress Before Pregnancy Reduce the Expression of Brain-Derived Neurotrophic Factor and N-Methyl-D-Aspartate Receptor in Hippocampus of Offspring Rats Associated with Impairment of Memory

To investigate the effect of stress before pregnancy on memory function and serum corticosterone (COR) levels, as well as the expression of brain-derived neurotrophic factor (BDNF), N-methyl-D-aspartate (NMDA) 2A (NR2A) and 2B (NR2B) receptors in the hippocampus of the offspring rats when they were 2 months postnatally. Adult female Sprague-Dawley (SD) rats were divided randomly into two groups: control group (n = 8) and chronic unpredictable stress (CUS) group (n = 12). All rats were tested in the open field test and sucrose intake test before and after CUS. The memory function of their offspring were tested in the Morris water maze. Serum COR levels were determined by using a standard radioimmunoassay kit. The expression of BDNF, NR2A and NR2B in the hippocampus of the offspring rats were studied by immunoreactivity quantitative analysis and real-time RT-PCR. (1) Following CUS, reduced open field test activity and decreased sucrose consumption were observed relative to controls. (2) The Morris water maze task demonstrated increased escape latency in the offspring rats of CUS group relative to controls (P < 0.01). No-platform probe testing showed reduced crossings for offspring of CUS relative to controls (P < 0.05). (3) CUS induced a significant increase in serum COR levels of the offspring rats (P < 0.01), but no difference was observed in the body or brain weight between the offspring of the two groups. (4) Immunoreactivity quantitative analysis shows that BDNF and NR2B in the offspring of CUS group was decreased in the CA3 and DG regions of the hippocampus compared to the control group offspring, but NR2A levels were not altered between the offspring of the two groups. (5) Real-time RT-PCR demonstrated that BDNF and NR2B mRNAs were significantly decreased in the offspring of the CUS group compared with the control group (P < 0.01). No significant difference in the levels of NR2A mRNA was detected between offspring of CUS and offspring of control groups. In our study, pregestational stress can increase serum corticosterone levels and reduce the expression of BDNF and NR2B in the hippocampus of offspring. These alterations are associated with impairment of memory in the adult offspring. These data suggest that, stress before pregnancy might have a profound influence on brain development of offspring, that may persist into and be manifested in adulthood.