人参皂苷Rd预处理对局灶性脑缺血再灌注大鼠NR2B受体和核酸内切酶G表达的影响

目的:观察人参皂苷Rd预处理对局灶性脑缺血再灌注大鼠基底节区N-甲基-D-天冬氨酸(NMDA)受体亚单位NR2B蛋白和核酸内切酶G(EndoG)表达变化的影响,探讨人参皂苷Rd治疗缺血性卒中的可能机制.方法:栓线法建立大鼠大脑中动脉闭塞模型,免疫组织化学染色和图像分析法检测局灶性脑缺血1 h再灌注1、6、24、72 h后基底节区NR2B和EndoG表达,评价人参皂苷Rd对NR2B和EndoG表达和脑梗死体积的影响.结果:缺血再灌注组缺血侧基底节区NR2B阳性表达显著增加,EndoG在细胞核内表达显著;再灌注不同时间点人参皂苷Rd处理组NR2B和EndoG阳性表达均显著降低(P<0.05或P<0.01),脑梗死体积显著缩小(P<0.01).结论:脑缺血再灌注后NMDA受体亚单位NR2B和凋亡诱导因子EndoG表达显著增加;人参皂苷Rd预处理能显著降低NR2B和EndoG表达,通过抑制兴奋性神经毒性和阻断神经细胞凋亡,缩小脑梗死体积,从而起神经保护作用.     

NMDA受体NR2B亚型基因对海马成年新生神经元形态发生的影响

目的研究NMDA受体NR2B亚型基因对海马成年新生颗粒细胞形态发生的影响。方法通过Cre-loxp重组酶系统构建NMDA受体NR2B亚型基因单细胞敲除模型。运用Neurolucida软件系统对野生型、NR2B基因敲除成年新生神经元进行形态重建,观察树突长度、树突复杂度以及棘突的变化。结果 NR2B基因敲除成年新生神经元外形与WT神经元相似,树突长度接近,棘突减少。Sholl分析显示在以胞体为中心,距胞体不同距离的树突亚区域内,树突复杂性(树突交叉)减低,与此同时棘突分布密度减低(P<0.05)。结论海马成年新生颗粒神经元NR2B基因敲除对新生细胞树突长度影响甚微,但降低了树突复杂性,减少棘突形成,从而导致了功能性整合入海马特定神经信息网络的障碍。     

二氮嗪预处理对Aβ_(1~42)作用神经元NR2B亚基蛋白表达的影响

目的研究ATP敏感的钾离子通道开放剂二氮嗪(diazoxide)预处理对Aβ1~42作用原代培养神经元N-甲基-D-天冬氨酸(NMDA)受体2B(NR2B)亚基蛋白表达的影响。方法原代培养大鼠皮层海马神经元并进行鉴定,将细胞随机分为对照组、单纯Aβ1~42干预组、二氮嗪预处理1h后Aβ1~42干预组(Aβ1~42+diazoxide)、单纯二氮嗪预处理组,并采用免疫印迹检测不同时间点(24 h、72 h)细胞NR2B亚基蛋白表达水平的变化。结果 Aβ1~42(2μmol/L)作用神经元24 h后,与对照组相比,单纯Aβ1~42干预组和Aβ1~42+diazoxide组的NR2B亚基蛋白表达均无明显改变。Aβ1~42作用神经元72 h后,与对照组比较,单纯Aβ1~42组NR2B亚基蛋白表达量显著升高(P0.05);而与单纯Aβ1~42干预组相比,Aβ1~42+diazoxide组NR2B亚基蛋白表达明显降低(P0.05)。结论 Aβ1~42作用原代培养神经元72 h,能够显著增加神经细胞NR2B亚基蛋白的表达量;同时,二氮嗪能拮抗Aβ1~42所引起的NR2B亚基蛋白表达量升高,提示二氮嗪可能通过NMDA受体通路影响Aβ1~42的细胞毒性作用。     

N-甲基-D-天冬氨酸受体NR2B亚基与神经元凋亡

神经元凋亡是缺血性卒中神经元死亡的重要形式之一.在神经元凋亡过程中,民N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体介导的兴奋性氨基酸毒性起着重要作用,是神经元凋亡的启动者和执行者.NR2B是该受体的主要调节亚基,其跨膜片段M2是一个面向胞质向膜内反折的膜襻区,形成离子通道的内壁,决定了NMDA受体离子通道对ca2+的通透性.谷氨酸主要通过钙超载介导细胞损伤,因此,NR2B亚基在缺血性卒中神经元凋亡中起着至关重要的作用.     

电针对脑缺血再灌注模型大鼠纹状体NMDA受体NR2亚基蛋白表达的影响

目的 观察脑缺血再灌注模型大鼠受损侧纹状体组织中NMDA受体调节亚单位NR2A和NR2B蛋白表达及其电针干预后的变化,探讨电针对缺血再灌注兴奋性氨基酸毒性的拮抗作用,进一步阐明电针治疗缺血性脑血管疾病的机制.方法 用数字随机表将15只SD大鼠随机分成3组:假手术组、模型组、电针组,每组各5只.采用改良Longa线栓法制作大脑中动脉短暂性缺血再灌注模型,电针组大鼠在再灌同时电针“百会”、“大椎”两穴(连续波,频率3 Hz,电流强度1～3 mA),30 min,深度均为10 mm.免疫组织化学法及图像处理系统检测分析大鼠受损侧纹状体NR2A、NR2B蛋白表达情况.结果 与假手术组相比,模型组大鼠纹状体组织中NR2A的表达量明显下降,而NR2B的表达量明显升高,给予电针“百会”、“大椎”穴后的大鼠纹状体组织NR2A表达细胞数及含量均明显高于模型组,表达NR2B的细胞数及含量均明显降低.结论 电针可能通过激活NR2A受体蛋白表达,抑制NR2B受体蛋白的过量表达,从而调节NMDA受体复合物的整体功能活性,减少NMDA受体介导的兴奋性氨基酸毒性反应,减轻脑缺血再灌注引起的局灶性脑损伤,发挥一定的脑神经保护作用.     

孤儿核受体NR4A与肺癌

NR4A核受体可被多种细胞外刺激因素激活而广泛参与调控生物的代谢、生长、分化、凋亡等过程.因NR4A核受体结构上的特点,其发挥转录活性不依赖配体,主要依赖转录后修饰.NR4A核受体参与肿瘤的调控是双向性的,既能促进肿瘤增殖,又能促进肿瘤细胞凋亡,与刺激因素、肿瘤类型、肿瘤所处的发展阶段及其在肿瘤细胞的亚细胞定位有关.NR4A核受体亚家族均参与肺癌发病机制,因此本文就NR4A核受体与肺癌的关系作一综述.     

NMDA受体NR1、NR2A/B在丘脑前核-海马CA1、CA3脑区和齿状回的分布与表达

目的 探讨NMDA受体NR1、NR2A/B在丘脑前核-海马CA1、CA3脑区和齿状回的分布与表达,以及丘脑前核-海马神经元的学习记忆功能及作用机制.方法 运用原位杂交检测技术观测丘脑前核及海马CA1、CA3和齿状回内NMDA受体NR1、NR2A 及NR2B mRNA的分布特点.结果 ①原位杂交阳性产物呈棕黄色,主要分布在神经元的胞浆中,胞核基本不着色.②在丘脑前核,阳性神经元分布较密集,细胞形态较一致.③在海马锥体层阳性神经元分布较多,呈带状.在分子层、多形层分布少.④NR1、NR2A/B在丘脑前核和海马CA1、CA3脑区及齿状回均有表达,其中NR1在齿状回表达水平最强,NR2B在丘脑前核、海马CA1、CA3和齿状回表达水平基本相同.结论 在丘脑前核-海马的局部神经元环路中NMDA受体NR1、NR2A及NR2B mRNA分布广泛.其中NR2B mRNA在丘脑前核和海马CA1、CA3脑区及齿状回表达水平基本相同,可能与此环路学习记忆有关.     

炎症后内脏高敏感大鼠前扣带回皮质NR2A、NR2B表达变化

背景:研究显示N-甲基-D-天冬氨酸(NMDA)受体参与了伤害性信号的传递,中枢前扣带回皮质(ACC)在内脏高敏感大鼠内脏疼痛反应的调节中起重要作用,该作用是由NMDA受体活性增强介导的。目的:检测炎症后内脏痛觉过敏大鼠ACC区域NMDA受体亚基NR2A、NR2B的表达变化,探讨两者在炎症后内脏高敏感形成中的作用。方法:以去氧胆酸结肠灌注建立炎症后内脏痛觉过敏大鼠模型。造模3周后观察实验组和对照组结肠组织病理学改变,以对结直肠扩张(CRD)的内脏运动反射(VMR)幅值为指标评价内脏痛敏感性的变化,以免疫荧光法和蛋白质印迹法检测ACC区域NR2A、NR2B表达情况。结果:实验组和对照组大鼠结肠组织均未见明显病理学改变。CRD压力为60 mm Hg(伤害性刺激)时,实验组VMR幅值显著高于对照组(P0.05)。与对照组相比,实验组ACC区域NR2A、NR2B荧光强度和蛋白表达量均显著上调(P0.05)。结论:ACC区域NMDA受体亚基NR2A、NR2B表达上调在炎症后内脏高敏感的形成中发挥重要作用。     

实验性氟中毒对SH-SY5Y细胞NMDA受体、Ca~(2+)及凋亡的影响

目的研究氟对SH-SY5Y细胞中NMDA受体、细胞内Ca~(2+)浓度及细胞凋亡的影响,探讨高氟蓄积对神经系统损伤的机制。方法体外培养SH-SY5Y细胞,加入终浓度为0.2 mmol/L和2 mmol/L的氟化钠,并用10μmol/L的NMDA受体拮抗剂MK801对染氟组进行干预。免疫荧光检测细胞内Ca~(2+)的浓度变化,流式细胞仪测定细胞凋亡率,Western blot法检测NMDA受体亚型NR1、NR2A、NR2B蛋白的表达。结果与对照组相比,高氟组中细胞凋亡率和细胞内Ca~(2+)平均荧光值明显增加,NR1和NR2A蛋白表达增加(P0.05)。对高氟组进行MK801干预后,细胞凋亡率和细胞内Ca~(2+)浓度减低(P0.05)。结论在SH-SY5Y细胞中NMDA受体亚型NR1和NR2A的过度表达参与了高氟诱导的神经细胞凋亡和坏死的过程,这种损伤机制可能是由于细胞内Ca~(2+)大量聚集所致。     

抗N-甲基-D天冬氨酸受体脑炎

正抗N-甲基-D天冬氨酸(N-Methyl-D-aspartate,NMDA)受体脑炎是近年来发现的一种自身免疫性脑炎,由NMDA受体NR1亚单位的Ig G抗体介导。以急性精神障碍、记忆障碍、癫痫、语言障碍等症状起病,之后进入反应低下状态,意识水平下降,通常伴有自主神经功能失调以及中枢性通气功能障碍。多见于年轻的成人与儿童,尤其是伴有卵巢畸胎瘤的女性。     

Metabotropic NMDA receptor function is required for β-amyloid–induced synaptic depression

The mechanisms by which β-amyloid (Aβ), a peptide fragment believed to contribute to Alzheimer’s disease, leads to synaptic deficits are not known. Here we find that elevated oligomeric Aβ requires ion flux-independent function of NMDA receptors (NMDARs) to produce synaptic depression. Aβ activates this metabotropic NMDAR function on GluN2B-containing NMDARs but not on those containing GluN2A. Furthermore, oligomeric Aβ leads to a selective loss of synaptic GluN2B responses, effecting a switch in subunit composition from GluN2B to GluN2A, a process normally observed during development. Our results suggest that conformational changes of the NMDAR, and not ion flow through its channel, are required for Aβ to produce synaptic depression and a switch in NMDAR composition. This Aβ-induced signaling mediated by alterations in GluN2B conformation may be a target for therapeutic intervention of Alzheimer’s disease.     

NR2B subunit of the NMDA glutamate receptor regulates appetite in the parabrachial nucleus

Diphtheria toxin-mediated, acute ablation of hypothalamic neurons expressing agouti-related protein (AgRP) in adult mice leads to anorexia and starvation within 7 d that is caused by hyperactivity of neurons within the parabrachial nucleus (PBN). Because NMDA glutamate receptors are involved in various synaptic plasticity-based behavioral modifications, we hypothesized that modulation of the NR2A and NR2B subunits of the NMDA receptor in PBN neurons could contribute to the anorexia phenotype. We observed by Western blot analyses that ablation of AgRP neurons results in enhanced expression of NR2B along with a modest suppression of NR2A. Interestingly, systemic administration of LiCl in a critical time window before AgRP neuron ablation abolished the anorectic response. LiCl treatment suppressed NR2B levels in the PBN and ameliorated the local Fos induction that is associated with anorexia. This protective role of LiCl on feeding was blunted in vagotomized mice. Chronic infusion of RO25-6981, a selective NR2B inhibitor, into the PBN recapitulated the role of LiCl in maintaining feeding after AgRP neuron ablation. We suggest that the accumulation of NR2B subunits in the PBN contributes to aphagia in response to AgRP neuron ablation and may be involved in other forms of anorexia.NMDA receptors form glutamate-gated ion channels that mediate many forms of synaptic plasticity under physiological conditions and neuronal death under excitotoxic pathological conditions (1, 2). NMDA receptors are tetrameric complexes composed of two obligatory NR1 subunits and two regulatory NR2 and/or NR3 subunits. Multiple subtypes of NMDA receptors are identified with distinct pharmacological and biophysical properties that are predominantly determined by the type of NR2 subunit (NR2A to NR2D). NR2A and NR2B subunits are the most abundant subunits in the adult brain and differ in channel properties, synaptic localization, and protein interaction elements, all of which can contribute to the induction of synaptic plasticity (3). Some recent studies suggest that NMDA receptors play a major role in regulating feeding behavior and energy homeostasis. For example, antagonism of hindbrain NMDA receptors increases food intake, whereas activation of NMDA receptors in the nucleus tractus solitarius (NTS) is necessary for cholecystokinin-induced reduction of food intake (4, 5). Injection of NMDA receptor agonists into the lateral hypothalamus elicits feeding in satiated animals, whereas NMDA blockade suppresses food intake and can reduce body weight (6). Genetic inactivation of NR1 subunits specifically in agouti-related protein (AgRP) neurons results in marked loss of food intake and body fat and impaired feeding after a fast (7). These findings implicate NMDA receptor signaling within several neuronal circuits in the control of feeding and energy balance.Emerging evidence suggests that a complex neural network including GABAergic AgRP neurons in the hypothalamic arcuate nucleus plays a pivotal role in the control of appetite and energy metabolism (8–10). Activation of AgRP neurons promotes food intake and body weight gain by inhibiting postsynaptic target neurons in the paraventricular hypothalamus (10). Ablation of AgRP neurons by administration of diphtheria toxin (DT) to mice that express the diphtheria toxin receptor (DTR) selectively in AgRP neurons (Agrp^DTR mice) results in aphagia and a fatal loss of body weight caused by the hyperactivity of postsynaptic neurons in the parabrachial nucleus (PBN) as a consequence of losing inhibitory GABAergic signals (11–13). This anorexic response can be prevented by benzodiazepine potentiation of GABA signaling, by genetic blockade of NMDA glutamate receptor signaling in the PBN, or by reducing glutamatergic input or output from the PBN, including knockdown of NMDA expression in the PBN (14). However, the physiological roles of NMDA receptors in the PBN in modulating appetitive behavior have not been identified.We explored the potential benefit of pretreatment with LiCl because this salt has been shown to affect the activity of the PBN and to modulate NMDA receptor functions. Lithium is commonly used to treat bipolar mood disorder, but it also can elicit robust neuroprotection against excitotoxicity in CNS neurons (15, 16). Long-term exposure to LiCl protects cultured cerebellar, cortical, and hippocampal neurons against glutamate-induced excitotoxicity; this protection can be attributed, in part, to the inhibition of NMDA receptor-mediated calcium influx (17). Significant weight gain is associated with prolonged lithium therapy in patients with bipolar disorder; these patients have enhanced preference for a high-calorie diet, but the mechanism(s) causing this metabolic effect are unknown (18). It is well documented that a single i.p. dose of LiCl produces gastrointestinal malaise that is coincident with the induction of Fos in certain brainstem structures, including the NTS and the PBN (19, 20). Exposure of rodents to a novel taste followed by LiCl treatment produces robust conditioned aversion to that taste, and electrolytic lesions of the PBN prevent LiCl-mediated conditioned taste aversion (21). These results are consistent with the idea that LiCl activates vagal afferents to the NTS, which then relays an excitatory, glutamatergic signal to the PBN where it induces Fos in a subpopulation of PBN neurons. We hypothesized that robust glutamatergic signaling induced in the PBN by LiCl treatment might lead to synaptic plasticity involving NMDA receptors and that these changes might influence the anorexia phenotype associated with the sudden loss of GABAergic input to the PBN from AgRP neurons.     

Duration of estrogen deprivation, not chronological age, prevents estrogen's ability to enhance hippocampal synaptic physiology

Whether estrogen replacement is beneficial to cognitive health is controversial. Some studies have shown that estrogen replacement therapy (ERT) relieves memory impairment associated with menopause in women, whereas others suggest that estrogen not only is incapable of providing a benefit, but actually can be detrimental. One possible explanation for this discrepancy in study findings could be the varying time after menopause at which ERT is initiated. It has been proposed that a critical period exists during which ERT must be administered to enhance cognitive function. This idea has yet to be tested directly using functional synaptic studies, however. Here we investigated whether prolonged hormone deprivation caused by ovariectomy (OVX) in young adult rats prevents the ability of estrogen replacement to increase synaptic function in the hippocampus to a degree necessary for estrogen-induced improvement in learning and memory. Remarkably, estrogen replacement was found to increase long-term potentiation, the current mediated by NR2B-containing NMDA receptors, and the dendritic spine density at CA3-CA1 synapses up to 15 months post-OVX. However, by 19 months post-OVX, the same estrogen replacement was unable to induce these changes. Importantly, this loss of estrogen's effectiveness was seen to be a consequence of the duration of deprivation. In female rats aged with their ovaries intact and examined at the same chronological age as the 19-month post-OVX group, estrogen replacement significantly increased synaptic function and spine density. These data clearly demonstrate that a critical period exists during which ERT must be administered, and that once this period passes, the beneficial effects are lost.     

Fyn kinase and NR2B-containing NMDA receptors regulate acute ethanol sensitivity but not ethanol intake or conditioned reward

BACKGROUND: The tyrosine kinase Fyn previously has been shown to play a key role in mediating acute tolerance to ethanol. Recently, we found that the compartmentalization of Fyn to the NR2B subunit of the NMDA receptor (NMDAR) in the hippocampus regulates Fyn phosphorylation of NR2B in response to ethanol, which mediates the acute tolerance of NMDAR to ethanol inhibition in hippocampal slices. In this study we determined, first, whether acute tolerance to ethanol inhibition is mediated via NR2B-containing NMDARs in vivo and, second, whether the increase in acute sensitivity to ethanol in the Fyn-/- mice influences ethanol consumption or ethanol's conditioned rewarding effects. METHODS: A loss of righting reflex test was used to study the acute/sedative effects of ethanol after intraperitoneal injections of sedative doses of ethanol. Conditioned place preference was used to study the rewarding properties of ethanol. The two-bottle choice protocol was used to measure oral ethanol self-administration and preference as described previously. RESULTS: We found that systemic injection of the NR2B-containing NMDAR selective antagonist, ifenprodil, abolished the differences between Fyn+/+ and Fyn-/- mice in sensitivity to the acute sedative effects of ethanol. Moreover, we found that Fyn-/- and Fyn+/+ mice did not differ in their voluntary ethanol consumption or in the rewarding properties of ethanol. CONCLUSIONS: Our results suggest that the interaction between Fyn and NR2B mediates the acute sedative effects of ethanol, and that alteration in acute ethanol sensitivity does not necessarily correlate with levels of ethanol consumption or the rewarding properties of ethanol.     

Specific involvement of postsynaptic GluN2B-containing NMDA receptors in the developmental elimination of corticospinal synapses

The GluN2B (GluRε2/NR2B) and GluN2A (GluRε1/NR2A) NMDA receptor (NMDAR) subtypes have been differentially implicated in activity-dependent synaptic plasticity. However, little is known about the respective contributions made by these two subtypes to developmental plasticity, in part because studies of GluN2B KO [Grin2b^−/− (2b^−/−)] mice are hampered by early neonatal mortality. We previously used in vitro slice cocultures of rodent cerebral cortex (Cx) and spinal cord (SpC) to show that corticospinal (CS) synapses, once present throughout the SpC, are eliminated from the ventral side during development in an NMDAR-dependent manner. To study subtype specificity of NMDAR in this developmental plasticity, we cocultured Cx and SpC slices derived from postnatal day 0 (P0) animals with different genotypes [2b^−/−, Grin2a^−/− (2a^−/−), or WT mice]. The distribution of CS synapses was studied electrophysiologically and with a voltage-sensitive dye. Synapse elimination on the ventral side was blocked in WT(Cx)-2b^−/−(SpC) pairs but not in WT(Cx)-2a^−/−(SpC) or 2b^−/−(Cx)-WT(SpC) pairs. CS axonal regression was also observed through live imaging of CS axons labeled with enhanced yellow fluorescent protein (EYFP) through exo utero electroporation. These findings suggest that postsynaptic GluN2B is selectively involved in CS synapse elimination. In addition, the elimination was not blocked in 2a^−/− SpC slices, where Ca²⁺ entry through GluN2B-mediated CS synaptic currents was reduced to the same level as in 2b^−/− slices, suggesting that the differential effect of GluN2B and GluN2A in CS synapse elimination might not be explained based solely on greater Ca²⁺ entry through GluN2B-containing channels.     

The genome of the nematode Pristionchus pacificus encodes putative homologs of RXR/Usp and EcR

Ecdysteroid signaling is an important regulator of arthropod development and reproduction. However, the role of ecdysteroid signaling in another Ecdysozoan animal, the nematode, remains unclear. We report here the identification, cloning, and temporal expression of genes encoding putative homologs of the two nuclear receptor components of the ecdysone receptor, RXR/Usp (NR2B) and EcR (NR1H), in the nematode Pristionchus pacificus. The P. pacificus genes Ppa-pnhr-1 and Ppa-pnhr-2 encode nuclear receptors with strong sequence similarity to RXR/Usp and EcR, respectively. Maximum likelihood analysis incorporating both DNA-binding and ligand-binding domains places the two proteins in the NR2B and NR1H groups with strong bootstrap support. RT-PCR analysis reveals that both Ppa-pnhr-1 and Ppa-pnhr-2 are expressed during larval development and that Ppa-pnhr-1 expression oscillates with the molting cycle. The identification of a putative ecdysone receptor in a nematode amenable to genetic analysis provides a powerful system to investigate the function and evolution of ecdysone receptor signaling in the Nematoda.