N-methyl-D-aspartate glutamate receptors (NMDARs) in stroke pathogenesis and treatments

N-methyl-D-aspartate glutamate receptors(NMDARs)play crucial roles in the pathogenesis of neuronal injuries following a stroke insult;therefore,a plethora of preclinical studies focus on better understanding functions of NMDARs and their associated signaling pathways.Over the past decades,NMDARs have been found to exert dual effects in neuronal deaths signaling and neuronal survival signaling during cerebral ischemia.Moreover,many complex intracellular signaling pathways downstream of NMDAR activation have been elucidated,which provide novel targets for developing much-needed neuro-protectants for patients with stroke.In this review,we will discuss the recent progress in understanding the underlying mechanisms of stroke related to NMDAR activation and the potential therapeutic strategies based on these discoveries.

Controlling N-methyl-D-aspartate receptor subunit 1 with calcitonin gene related peptide after cerebral ischemic injury

BACKGROUND: Activation of N-methyl-D-aspartate receptor (NMDAR) is a key link of exitotoxicity at the phase of cerebral ischemic injury. Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid receptors, over-excitation can cause neuronal apoptosis. Calcitonin gene related peptide has a strongly biological activity. On one hand, it can protect ischemic neurons through inhibiting the expression of NMDAR1 mRNA; on the other hand, it can play the protective effect through down-regulating the expression of NMDAR1 mRNA by exogenous calcitonin gene related peptide. OBJECTIVE: To observe the expression of NMDAR1 and the regulatory effect of calcitonin gene related peptide on the expression of NMDAR1 mRNA and protein in the cerebral cortex of rats with focal cerebral ischemia/reperfusion (I/R). DESIGN: Randomized controlled animal study. SETTING: China Medical University. MATERIALS: A total of 216 healthy male Wistar rats, general grade, weighing 250-280 g, were selected in this study. Twelve rats were randomly selected to regard as control group; meanwhile, other 204 rats were used to establish middle cerebral artery occlusion/reperfusion (MACO) models. The main reagents were detailed as follows: calcitonin gene related peptide (Sigma Company); calcitonin gene related peptide kit (Boster Company); antibody Ⅰ, Ⅱ and antibody β-actin Ⅰ, Ⅱ of NMDAR1 mRNA and chemiluminescence reagent (Santa Cruz Company, USA). METHODS: The experiment was carried out in the Laboratory of Neurobiology of China Medical University from August 2005 to June 2006. ① Right MCAO models of rats were established to cause focal ischemia and scored based on Zea Longa five-grade scale. If the scores were 1, 2 and 3 after wakefulness, the MACO models were established successfully and involved in the experiment. A total of 120 rats with successful modeling were randomly divided into I/R group and administration group with 60 in each group. All rats in the both groups were observed at five time points, including 6, 12, 24, 48 and 72 hours after reperfusion and after 2-hour ischemia, with 12 experimental animals at each time point. Six rats were prepared for detection of hybridization in situ, and the other 6 were used for Western blotting histochemical detection. Rats in the control group were opened their skin to separate common carotid artery and not treated with line and drugs. In addition, rats in the I/R group were treated with 1 mL saline at 2 hours after focal cerebral ischemia, and then, rats in the administration group were treated with 1 mL (1 g/L) calcitonin gene related peptide at 2 hours after focal cerebral ischemia. ② The expression of NMDAR1 mRNA was detected with hybridization in situ at various time points; moreover, the expression of NMDAR1 protein was measured with Western blotting method at various time points. The results were analyzed with Metamoph imaging analytical system. MAIN OUTCOME MEASURES: The expression of NMDAR1 mRNA and its protein in cortical neurons of rats at various time points. RESULTS: A total of 84 rats were excluded because of non-symptoms, exanimation or death; and then, 132 rats were involved in the final analysis. The expression of NMDAR1 mRNA and its protein in cortical neurons of rats in the control group was 0.205±0.001 and 0.184±0.001, respectively; after I/R, expression of NMDAR1 mRNA and its protein was up-regulated, especially, expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.245±0.003, 0.287±0.004, 0.354±0.008, 0.284±0.002 and 0.217±0.006, respectively; moreover, expression of protein at 6, 12, 24, 48 and 72 hours was 0.222±0.003, 0.261±0.028, 0.311±0.004, 0.259±0.013 and 0.210±0.008, respectively. There was significant difference between the two groups (0.205±0.001, P < 0.01). The expression was up-related in the former 24 hours, reached peak at 24 hours, down-regulated, and decreased to the level of control group at 72 hours. Except 72 hours, the expression of NMDAR1 mRNA and its protein was lower in administration group than that in I/R group at other four time points. In addition, the expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.223±0.005, 0.243±0.001, 0.292±0.002, 0.250±0.003 and 0.213±0.003, respectively; moreover, the expression of protein at 6, 12, 24, 48 and 72 hours was 0.216±0.006, 0.245±0.025, 0.276±0.003, 0.241±0.045 and 0.202±0.013, respectively. There was significant difference at various time points (P < 0.05). CONCLUSION: The expressions of NMDAR1 mRNA and its protein of peripheral cortical neurons are up-related in ischemic area after focal cerebral I/R. Meanwhile, exogenous calcitonin gene related peptide can protect cortical neurons through inhibiting expression of NMDAR1 mRNA and its protein after focal cerebral I/R.

脑络欣通对脑缺血再灌大鼠神经细胞凋亡率及NMDAR蛋白表达的影响

目的:研究脑络欣通对气虚血瘀脑缺血再灌注模型鼠神经细胞凋亡率及N-甲基-D-天冬氨酸受体(NMDAR)蛋白表达的影响。方法:采用饥饿、劳累、高脂饮食等多因素复制大鼠气虚血瘀模型,然后用线栓法阻断大鼠左侧大脑中动脉2h再灌注6,24,72h,复制局灶性脑缺血再灌注模型,用流式细胞仪和免疫组化法分别检测缺血半暗带神经细胞凋亡率和NMDAR蛋白表达。结果:与模型组比较,脑络欣通组神经细胞凋亡率和NMDAR蛋白表达显著减少(P<0.01)。结论:脑络欣通抑制NMDAR蛋白表达,是其抗脑缺血再灌注后神经细胞凋亡的机制之一。

弓状核损毁模型大鼠海马组织中GABA_A受体、NMDAR_1受体、c-fos蛋白、NGF受体的表达

目的 :探讨谷氨酸钠造成的下丘脑弓状核损毁模型鼠海马组织中γ -氨基丁酸 (GABAA)受体、N -甲基-D -天冬氨酸受体 (NMDAR1 )、c -fos蛋白、神经生长因子 (NGF)受体的表达。方法 :皮下注射谷氨酸钠建立SD新生大鼠弓状核损毁模型。正常组注射等量生理盐水作对照。GABAA 受体、NMDAR1 受体、c -fos蛋白、NGF受体的表达以免疫组化染色显示。结果 :造模组大鼠海马组织中GABAA 受体、NMDAR1 受体的表达均明显低于正常组 ,P<0 .0 5 ,NGF受体的表达明显高于正常组 ,P <0 .0 5 ,c-fos蛋白表达略高于正常组 ,但差异无统计学意义 ,P >0 .1。结论 :谷氨酸钠损毁弓状核对大鼠海马组织中GABAA 受体、NMDAR1 受体、NGF受体的表达有明显影响。

Chinese Tuina remodels the synaptic structure in neuropathic pain rats by downregulating the expression of N-methyl D-aspartate receptor subtype 2B and postsynaptic density protein-95 in the spinal cord dorsal horn

OBJECTIVE:To investigate whether the Chinese massage system,Tuina,exerts analgesic effects in a rat model of chronic constriction injury(CCI)by remodeling the synaptic structure in the spinal cord dorsal horn(SCDH).METHODS:Sixty-nine male Sprague–Dawley rats were randomly and evenly divided into the normal group,sham group,CCI group,CCI+Tuina group,CCI+MK-801[an N-methyl D-aspartate receptor subtype 2B(NR2B)antagonist]group,and CCI+MK-801+Tuina group.The neuropathic pain model was established using CCI with right sciatic nerve ligation.Tuina was administered 4 d after CCI surgery,using pressing manipulation for 10 min,once daily.Motor function was observed with the inclined plate test,and pain behaviors were observed by the Von Frey test and acetone spray test.At 19 d after surgery,the L3-L5 spinal cord segments were removed.Glutamate,interleukin 1β(IL-1β),and tumor necrosis factor-α(TNF-α)levels were detected by enzyme-linked immunosorbent assay.The protein expression levels of NR2B and postsynaptic density protein-95(PSD-95)were detected by Western blot,and the synaptic structure was observed by transmission electron microscopy(TEM).RESULTS:CCI reduced motor function and caused mechanical and cold allodynia in rats,increased glutamate concentration and TNF-αand IL-1βlevels,and increased expression of synapse-related proteins NR2B and PSD-95 in the SCDH.TEM revealed that the synaptic structure of SCDH neurons was altered.Most of these disease-induced changes were reversed by Tuina and intrathecal injection of MK-801(P<0.05 or<0.01).For the majority of experiments,no significant differences were found between the CCI+MK-801 and CCI+MK-801+Tuina groups.CONCLUSIONS:Chinese Tuina can alleviate pain by remodeling the synaptic structure,and NR2B and PSD-95 receptors in the SCDH may be among its targets.

基于cAMP/PKA/NMDAR信号通路探讨黑逍遥散对APP/PS1双转基因AD小鼠海马神经元突触可塑性的影响

目的:探究黑逍遥散通过调控环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/N-甲基-D-天冬氨酸受体(NMDAR)信号通路干预APP/PS1小鼠突触可塑性的作用及其机制。方法:选取4月龄雄性C57BL/6J小鼠12只作空白组,同月龄雄性APP/PS1双转基因小鼠60只,随机分为模型组、KW-6002(腺苷受体的拮抗剂)组(3 mg·kg^(-1))及黑逍遥散高、中、低剂量组(22.10、11.05、5.53 g·kg^(-1)),每组12只,对应药物连续干预90 d。透射电镜观察海马神经元突触超微结构,高尔基染色观察海马CA1区神经元树突棘密度,蛋白免疫印迹法(Western blot)检测cAMP、PKA、N-甲基-D-天冬氨酸受体1(NR1)、N-甲基-D-天冬氨酸受体2A(NR2A)、N-甲基-D-天冬氨酸受体2B(NR2B)、突触后致密物质95(PSD95)、突触蛋白1(SYN1)的蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测cAMP、PKA、NR1的mRNA表达,酶联免疫吸附测定法(ELISA)检测小鼠海马白细胞介素-12(IL-12)、白细胞介素-4(IL-4)含量。结果:与空白组比较,模型组小鼠海马CA1区突触前膜与突触后膜分界线清晰程度欠佳,突触囊泡明显减少且分布散乱,突触后膜密度降低,海马CA1区神经元树突棘形态欠规整,排列不连续、密度减少(P<0.01),海马cAMP、PKA、NR1、NR2A、NR2B、PSD95、SYN1的蛋白表达降低(P<0.01),cAMP、PKA、NR1的mRNA表达下调(P<0.01),海马IL-12含量增多、IL-4含量减少(P<0.01);与模型组比较,各用药组小鼠海马CA1区突触前膜与后膜分界线清晰明了且结构完整,突触囊泡增多,排列密集,突触后膜密度增加,神经元树突棘形态较为规则,排列连续,成熟型树突棘密度增多(P<0.05,P<0.01),海马cAMP、PKA、NR1、NR2A、NR2B、PSD95、SYN1的蛋白表达增加(P<0.05,P<0.01),cAMP、PKA、NR1的mRNA表达提高(P<0.01),海马IL-12含量降低(P<0.01),IL-4含量增加(P<0.01)。结论:黑逍遥散能改善APP/PS1小鼠海马神经元结构形态,其作用机制可能与激活cAMP/PKA/NMDAR信号通路、修复突触可塑性有关。

N-methyl-D-aspartic acid receptor 1 （NMDAR1） aggravates secondary inflammatory damage induced by hemin-NLRP3 pathway after intracerebral hemorrhage

Objective： Inflammation plays a critical role in secondary brain damage after intracerebral hemorrhage （ICH）. However, the mechanisms of inflammatory injury following ICH are still unclear, particularly the involvement of NLRP3 inflammasome, which are crucial to sterile inflammatory responses. In this study, we aim to test the hypothesis that NLRP3 signaling pathway takes a vital position in ICH-induced sec- ondary inflammatory damage and detect the role of N-methyl-D-aspartic acid receptor 1 （NMDARI） in this progress. Methods： ICH was induced in mice by microinjection of heroin into the striatum. The protein levels of NMDAR1, NMDAR1 phosphorylation, NLRP3 and IL-113 were measured by Western blot. The binding of NMDARI to NLRP3 was detected by immunoprecipitation. Results： The expression of NMDARI, NMDAR1 phosphorylation, NLRP3 and IL-I ~ were rapidly increased after ICH. Heroin treatment enhanced NMDAR1 expression and NMDAR1 phosphorylation, as well in cultured microglial cells treated by hemin. Hemin up-regulated NLRP3 and IL-I]3 level, which was reversed by MK801 （NMDAR antagonist） in vitro. Hemin also promoted the binding of NMDAR1 to NLRP3. Conclusion： Our findings suggest that NMDARI plays a pivotal role in hemin-induced NLRP3-mediated inflammatory damage through synergistic activation.

阿尔茨海默病大鼠海马N-甲基天冬氨酸受体和丝裂酶原激活蛋白激酶的表达

目的探讨N甲基天冬氨酸受体(NMDAR)及丝裂酶原激活蛋白激酶(MAPK)在阿尔茨海默病(AD)发病中的变化及其可能机制。方法将β淀粉样肽(Aβ1～40)1μl(10g L)在立体定位仪下注入大鼠海马建立大鼠AD模型。2周后测水迷宫潜伏期、长时程增强(LTP)、原位杂交方法检测大鼠海马NMDAR mRNA的表达及免疫组织化学SABC法检测MAPK的蛋白表达,并应用显微图像分析系统对两者的表达进行分析。结果AD模型组Aβ注射2周后,水迷宫潜伏期与模型组术前及对照组相比显著延长(P<0.05);模型组刺激前后fEPSP斜率变化及高频刺激增加的幅值与对照组相比显著下降(P<0.01);模型组海马CA1区、CA3区NMDAR mRNA及MAPK蛋白的表达显著降低(P<0.05或P<0.01)。相关性分析表明,AD模型2周后LTP检测的结果与NMDAR mRNA的表达及MAPK的蛋白表达呈正相关。结论Aβ通过抑制MAPK信号通路和降低NMDA受体磷酸化状态,导致大鼠海马LTP降低和学习记忆功能减低,参与AD学习记忆障碍和痴呆形成。

Study on the neurotoxic effects of low-level lead exposure in rats

Objective: To investigate effects of developmental lead exposure on nitric oxide synthase (NOS) activity in different brain regions and on N-methyl-D-aspartate (NMDA) receptor mRNA expression in the hippocampus of rats. On the basis of these observations, we explored possible mechanisms by which lead exposure leads to impaired learning and memorizing abilities in children. Methods: A series of rat animal models exposed to low levels of lead during the developing period was established (drinking water containing 0.025%, 0.05% and 0.075% lead acetate). NOS activities in the hippocampus, the cerebral cortex, the cerebellum and the brain stem were determined with fluorescence measurement and levels of mRNA expression of the NMDA receptor 2A (NR2A) subunit and NMDA receptor 2B (NR2B) subunit in the rat hippocampus were measured with Retro-translation (RT-PCR). Results: There were no differences in the body weight of rat pups between any of the groups at any given time (P>0.05). The blood lead level of Pb-exposed rat pups showed a systematic pattern of change: at 14 d of age, it was lower than that at 7 d of age, then rising to the peak level at 21 d and finally falling to lower levels at 28 d. The hippocampal NOS activities of lead-exposed groups were all lower than that of the control group on the 21 st and 28th day (P<0.01). NOS activities in the cerebellum of lead-exposed groups were all lower than that of the control group on the 21 st and 28th day (P<0.001) and the NOS activity of the 0.025% group was significantly lower than that of the 0.05% and 0.075% groups on the 28th day (P<0.05).NOS activity in the cerebral cortex of the 0.075% group was significantly lower than that of the control, 0.025% and 0.05% groups on the four day spans (P<0.001). There was no significant difference of NOS activity in the brain stem between any lead-exposed group and the control group on the four day spans. In the 0.05% and the 0.075% groups, the level of NR2A mRNA expression was higher than that in the control group at 7 d and 14 d of age (P<0.05). In the 0.025% group, the level of NR2A was found to be higher than that in the control group at 7 d of age only (P<0.05). No significant differences were found for the levels of NR2B mRNA expression between any of the groups at any given time. Conclusions: NOS activity in the hippocampus, the cerebral cortex and the cerebellum are inhibited by lead exposure. The degree of the inhibitory effect depends on the time span of exposure and the lead concentration. Developmental low-level lead exposure was found to raise the level of NR2A mRNA expression in the hippocampus of rats. Developmental low-level lead exposure does not affect the level of NR2B mRNA expression in the hippocampus.

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABA_A receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and IMUS), respectively in the Mg2+-free external solution containing 1 μmol/L glycine at a holding potential (VH) of -40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 μmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 nmol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2+ (10 μmol/L) or La3+(30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of IGABA by NMDA application; (iii) the suppression of IGABA by NMDA was inhibited by KN-62, a cal-cium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition.

GLYX-13 pretreatment ameliorates long-term isoflurane exposure-induced cognitive impairment in mice

Accumulating evidence indicates that inhalation anesthetics induce or increase the risk of cognitive impairment. GLYX-13（rapastinel） acts on the glycine site of N-methyl-D-aspartate receptors（NMDARs） and has been shown to enhance hippocampus-dependent learning and memory function. However, the mechanisms by which GLYX-13 affects learning and memory function are still unclear. In this study, we investigated these mechanisms in a mouse model of long-term anesthesia exposure. Mice were intravenously administered 1 mg/kg GLYX-13 at 2 hours before isoflurane exposure（1.5% for 6 hours）. Cognitive function was assessed using the contextual fear conditioning test and the novel object recognition test. The mRNA expression and phosphorylated protein levels of NMDAR pathway components, N-methyl-D-aspartate receptor subunit 2B（NR2B）-Ca2+/calmodulin dependent protein kinase II（CaMKII）-cyclic adenosine monophosphate response element binding protein（CREB）, in the hippocampus were evaluated by quantitative RT-PCR and western blot assay. Pretreatment with GLYX-13 ameliorated isoflurane exposure-induced cognitive impairment and restored NR2B, CaMKII and CREB mRNA and phosphorylated protein levels. Intracerebroventricular injection of KN93, a selective CaMKII inhibitor, significantly diminished the effect of GLYX-13 on cognitive function and NR2B, CaMKII and CREB levels in the hippocampus. Taken together, our findings suggest that GLYX-13 pretreatment alleviates isoflurane-induced cognitive dysfunction by protecting against perturbation of the NR2B/CaMKII/CREB signaling pathway in the hippocampus. Therefore, GLYX-13 may have therapeutic potential for the treatment of anesthesia-induced cognitive dysfunction. This study was approved by the Experimental Animal Ethics Committee of Drum Tower Hospital affiliated to the Medical College of Nanjing University, China（approval No. 20171102） on November 20, 2017.

Protective effects of Bushen Tiansui decoction on hippocampal synapses in a rat model of Alzheimer's disease

Bushen Tiansui decoction is composed of six traditional Chinese medicines：Herba Epimedii,Radix Polygoni multiflori,Plastrum testudinis,Fossilia Ossis Mastodi,Radix Polygalae,and Rhizoma Acorus tatarinowii.Because Bushen Tiansui decoction is effective against amyloid beta（Aβ） toxicity,we hypothesized that it would reduce hippocampal synaptic damage and improve cognitive function in Alzheimer＇s disease.To test this hypothesis,we used a previously established animal model of Alzheimer＇s disease,that is,microinjection of aggregated Aβ25–35 into the bilateral brain ventricles of Sprague-Dawley rats.We found that long-term（28 days） oral administration of Bushen Tiansui decoction（0.563,1.688,and 3.375 g/m L;4 m L/day） prevented synaptic loss in the hippocampus and increased the expression levels of synaptic proteins,including postsynaptic density protein 95,the N-methyl-D-aspartate receptor 2 B subunit,and Shank1.These results suggested that Bushen Tiansui decoction can protect synapses by maintaining the expression of these synaptic proteins.Bushen Tiansui decoction also ameliorated measures reflecting spatial learning and memory deficits that were observed in the Morris water maze（i.e.,increased the number of platform crossings and the amount of time spent in the target quadrant and decreased escape latency） following intraventricular injections of aggregated Aβ25–35 compared with those measures in untreated Aβ_（25–35）-injected rats.Overall,these results provided evidence that further studies on the prevention and treatment of dementia with this traditional Chinese medicine are warranted.

电压门控钙通道辅助亚基α2δ-1的结构和分子药理

电压门控钙通道(voltage-gated calcium channel, VGCC)辅助亚基α2δ-1在神经元兴奋传递中起促进作用,是加巴喷丁(gabapentin)和普瑞巴林(pregabalin)的作用靶点。α2δ-1由高度糖基化的α2亚基和δ亚基以4对二硫键相连,包含4个串联的Cache结构域和1个von Willebrand A结构域,但δ亚基的C-末端结构尚未完全解析。α2δ-1的结构和作用机制研究进展主要聚焦于以下3点:(1)α2δ-1的C-末端,传统观点认为含有跨膜基序(motif)。另一个观点认为是GPI锚定,尚缺乏直接的实验证明;(2)α2δ-1对电压门控钙离子通道的影响,包括直接的和非直接的调控对电压门控钙通道的膜上转运和电流的影响,以及α2δ-1的翻译后修饰对钙电流的影响;(3)α2δ-1不依赖于钙通道,而与其他蛋白质存在相互作用,互作效应表现在兴奋性电流增大或促进神经突触发生。在疾病研究方面,本文主要综述α2δ-1在慢性疼痛中的机制研究。α2δ-1参与的生理活动的分子机制复杂,现已突破传统的局限于电压门控钙通道辅助亚基的角色,并将有可能通过α2δ-1建立跨细胞膜的蛋白质-蛋白质相互作用网络的动态膜微区,以进一步评估其生理、病理和药理的相关性。

Activation of glycine site and GluN2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats:Implications for affective pain

Objective The rostral anterior cingulate cortex （rACC） is implicated in processing the emotional component of pain. N-methyl-D-aspartate receptors （NMDARs） are highly expressed in the rACC and mediate painrelated affect by activating a signaling pathway that involves cyclic adenosine monophosphate （cAMP）/protein ki- nase A （PKA） and/or extracellular regulated kinase （ERK）/cAMP-response element-binding protein （CREB）. The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC. Methods Immunohistochemistry and Western blot analy- sis were used to separately assess the expression of phospho-ERK （pERK） and phospho-CREB （pCREB） in vitro and in vivo. Double immunostaining was also used to determine the colocalization of pERK and pCREB. Results Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC, which was inhibited by the NMDAR antago- nist DL-2-amino-5-phospho-novaleric acid. Selective blockade of the NMDAR GluN2B subunit and the glycine- binding site, or degradation of endogenous D-serine, a co-agonist for the glycine site, significantly decreased the up- regulation of pERK and pCREB expression in the rACC. Further, the activated ERK predominantly colocalized with CREB. Conclusion Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats, and these might be fundamental molecular mechanisms underlying pain affect.

Knock-down of postsynaptic density protein 95 expression by antisense oligonucleotides protects against apoptosis-like cell death induced by oxygen-glucose deprivation in vitro

Objective Postsynaptic density protein 95 （PSD-95） plays important roles in the regulation of glutamate signaling, such as that of N-methyl-D-aspartate receptors （NMDARs）. In this study, the functional roles of PSD-95 in tyrosine phosphorylafion of NMDAR subunit 2A （NR2A） and in apoptosis-like cell death induced by oxygen-glucose de- privation （OGD） in cultured rat cortical neurons were investigated. Methods We used immunoprecipitation and immuno- blotting to detect PSD-95 protein level, tyrosine phosphorylation level of NR2A, and the interaction between PSD-95 and NR2A or Src. Apoptosis-like cells were observed by 4,6-diamidino-2-phenylindole staining. Results Tyrosine phospho- rylation of NR2A and apoptosis-like cell death were increased after recovery following 60-min OGD. The increases were attenuated by pretreatment with antisense oligonucleotides against PSD-95 before OGD, but not by missense oligonucle- otides or vehicle. PSD-95 antisense oligonucleotides also inhibited the increased interaction between PSD-95 and NR2A or Src, while NR2A expression did not change under this condition. Conclusion PSD-95 may be involved in regulating NR2A tyrosine phosphorylation by Src kinase. Inhibition of PSD-95 expression can be neuroprotective against apoptosis- like cell death after recovery from OGD.