靶向抗原递呈细胞激活Flk1重组减毒鼠伤寒沙门菌的构建及其抑制树突状细胞凋亡的初步研究

目的 观察靶向抗原递呈细胞激活血管内皮生长因子受体-2(Flk1)的重组减毒鼠伤寒沙门菌对树突状细胞凋亡的抑制作用.方法 构建含Flk1与靶向抗凋亡基因BAK siRNA的重组质粒载体pFlk1-U6BAK,将其转化至减毒鼠伤寒沙门菌株中,携带pFlk1-U6BAK和pFlk1的重组减毒鼠伤寒沙门菌分别感染经体外培养的小鼠树突状细胞;Western blotting法检测树突状细胞Flk和BAK表达水平,MTT法检测树突状细胞增殖活性.结果 成功构建了含Flk1和靶向抗凋亡基因BAK siRNA的重组质粒载体pFlk1-U6BAK.经携带pFlk1-U6BAK的重组减毒鼠伤寒沙门菌感染后,树突状细胞在稳定表达Flk的同时,BAK表达水平显著降低;而经携带pFlk1-U6BAK的重组减毒鼠伤寒沙门菌感染后,树突状细胞增殖活性明显升高.结论 携带pFlk1-U6BAK的重组减毒鼠伤寒沙门菌可以在稳定表达Flk的同时,通过下调凋亡基因BAK表达水平而抑制树突状细胞凋亡,有望成为较为有效的抗肿瘤血管生成疫苗.     

Nurrl基因在大鼠体外培养骨髓基质细胞的表达

目的：为获得高效表达孤儿核受体(orphan nuclear receptor，Nurrl)基因的骨髓基质细胞。方法：采用分子克隆技术，构建携带Nurrl基因的重组腺相关病毒(adeno-associated virus，AAV)载体；与包装质粒pAAV-RC和辅助质粒phelper一起用磷酸钙法转染包装细胞HEK293制备具有感染活性的AAV-Nurrl病毒粒子；用病毒上清液感染原代培养的大鼠骨髓基质细胞(Marrow stromal cells，MSCs)，并用免疫细胞化学方法检测阳性细胞。结果：经酶切鉴定和DNA测序，得到了序列正确的重组pAAV-Nurr1；感染HTl080细胞进行病毒滴度测定，每mL病毒贮存液可达10^12个阳性细胞；获得了Nurrl阳性的骨髓基质细胞，阳性率在60％以上。结论：重组AAV携带的Nurrl基因能够在MSCs中表达，本文为进一步探讨将该细胞用于帕金森病基因治疗的可能性研究奠定了基础。     

Neurturin基因在体外培养的大鼠骨髓基质细胞中的表达

目的构建携带一种神经营养因子Neurturin(NTN)基因的腺病毒(Ad),使其在骨髓基质细胞(BMSCs)中表达。方法采用分子克隆技术,构建携带NTN基因的Ad载体,用脂质体法转染、包装HEK293细胞,制备具有感染活性的Ad-NTN病毒粒子;用病毒上清液感染原代培养的大鼠BMSCs,并用免疫细胞化学方法检测阳性细胞。用W estern B lot检测转染Ad-NTN的BMSCs细胞上清液。结果经酶切鉴定和DNA测序,得到重组Ad-NTN,对被感染的HEK293细胞进行病毒滴度测定,每毫升病毒贮存液可达1×108.5半数组织培养感染量(TC ID50);将病毒贮存液感染BMSCs,获得了表达NTN的BMSCs,阳性率为65%。W estern B lot检测证实BMSCs上清液中出现特异性NTN条带。结论重组Ad携带的NTN基因能够在BMSCs中表达。     

大鼠GLUT3基因重组腺病毒载体的构建及鉴定

目的构建携带大鼠葡萄糖转运体3（GLUT3）基因的复制缺陷型重组腺病毒载体，为研究GLUT3的生物学功能提供工具。方法采用逆转录-聚合酶链反应（RT-PCR）的方法获取大鼠GLUT3基因全长cDNA片段，将其克隆至穿梭质粒pShuttle中构建穿梭质粒pShuttle-GLUT3，经酶切后与线性化的腺病毒骨架质粒pAdeno-X体外连接并转化大肠杆菌DH5α，构建重组腺病毒质粒pAd-GLUT3，酶切线性化重组腺病毒质粒后转染293细胞包装成重组病毒颗粒，重组腺病毒在293细胞中反复扩增数代后，分别用聚合酶链反应（PCR）及免疫印记法（western blot）的方法从基因和蛋白表达水平鉴定重组的腺病毒。结果经DNA测序和PCR分析显示GLUT3 cDNA序列正确。成功筛选出重组腺病毒质粒pAd-GLUT3后在HEK293细胞中成功包装出重组病毒，包装后冻融细胞行PCR及western blot检测表明重组腺病毒包装成功。结论本研究成功构建了携带大鼠GLUT3基因的复制缺陷型重组腺病毒载体。     

Nurrl基因在大鼠体外培养骨髓基质细胞的表达

目的:为获得高效表达孤儿核受体(orphan nuclear receptor,Nurrl)基因的骨髓基质细胞.方法:采用分子克隆技术,构建携带Nurrl基因的重组腺相关病毒(adeno-associated virus,AAV)载体;与包装质粒pAAV-RC和辅助质粒phelper一起用磷酸钙法转染包装细胞HEK293制备具有感染活性的AAV-Nurrl病毒粒子;用病毒上清液感染原代培养的大鼠骨髓基质细胞(Marrow stromal cells,MSCs),并用免疫细胞化学方法检测阳性细胞.结果:经酶切鉴定和DNA测序,得到了序列正确的重组pAAV-Nurrl;感染HT1080细胞进行病毒滴度测定,每mL病毒贮存液可达1012个阳性细胞;获得了Nurrl阳性的骨髓基质细胞,阳性率在60%以上.结论:重组AAV携带的Nurrl基因能够在MSCs中表达,本文为进一步探讨将该细胞用于帕金森病基因治疗的可能性研究奠定了基础.     

以海马神经元为靶细胞Ad-Easy系统快速构建携带PTEN基因的重组腺病毒表达载体

摘要：目的：应用Ad-Easy腺病毒载体系统快速构建携带PTEN (Phosphatase and tensin homolog deleted on chromosome ten)基因的重组腺病毒表达载体,为应用基因技术研究PTEN对缺血性脑损伤后的神经保护作用奠定基础。方法：采用RT-PCR法从大鼠海马神经元扩增获得目的基因PTEN, 克隆入pAdTrack-CMV穿梭质粒（含绿色荧光蛋白（Green fluorescence protein,GFP）基因）形成转移载体pAdTrack-CMV-PTEN,将其在大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy-1同源重组;重组子酶切鉴定正确后转染HEK293细胞,PCR分析鉴定扩增情况及Western blot检测受腺病毒感染的海马神经元内PTEN蛋白的表达。结果：荧光显微镜检测到受腺病毒感染的HEK293细胞表达GFP,出现明显的细胞病变效应（Cytopathic effect,CPE）,经3轮扩增得到了病毒所需滴度。PTEN蛋白在受感染腺病毒神经元内表达显著增强。结论：利用Ad-Easy系统成功快速的构建了携带PTEN基因的腺病毒表达载体,为研究基因治疗缺血性脑损伤奠定了基础。     

野生型Ⅱ型神经纤维瘤病基因Ⅱ亚型真核表达载体的构建及其功能

目的构建野生型Ⅱ型神经纤维瘤病基因（NF2）Ⅱ亚型的真核表达载体，观察其在人胚胎肾细胞293（HEK293）中的表达并检测其转染神经鞘瘤细胞（RT4）后的功能。方法以人胎脑cDNA文库中的NF2Ⅱ亚型基因为模板．应用聚合酶链反应技术扩增野生型NF2Ⅱ亚型基因，并定向克隆至真核表达载体pEGFP-N1质粒中：经鉴定正确的重组质粒pEGFP-NF2Ⅱ亚型，用Lipofectamine2000脂质体导人人胚胎肾细胞293，通过荧光显微镜和Western Blotting法观察基因表达水平：随后转染大鼠神经鞘瘤细胞．利用水溶性四氮唑法观察野生型NF2Ⅱ亚型基因对大鼠神经鞘瘤细胞增殖的影响。结果经序列分析证实，克隆的NF2Ⅱ亚型基因与Genebank中的序列完全一致。DNA琼脂糖凝胶电泳检测显示，经双酶切后的质粒pEGFPcDNA片段大小约为4700bp：NF2Ⅱ亚型基因cDNA片段大小为l770bp，NF2Ⅱ亚型基因的真核表达载体可瞬时转染人胚胎肾细胞293，通过荧光显微镜和Westem Blotting方法得到证实；水溶性四氮唑法检测显示，转染pEGFP-NF2Ⅱ亚型并表达NF2Ⅱ亚型编码蛋白的293细胞与对照组293细胞的增殖指数相同。结论所构建的野生型NF2Ⅱ亚型基因真核表达载体可在人胚胎肾细胞293中表达，且不具有抑制肿瘤细胞增殖的特性。     

携带低氧诱导因子1αmu和人源化海肾绿色荧光蛋白双基因真核表达载体 构建及其在HEK293A细胞中的表达*

背景：低氧诱导因子1能够调控多种基因共同表达,在骨缺损部位可诱导成熟的血管生成,为各种细胞的成骨分化和成骨活动提供营养支持和代谢保证,促进骨愈合,但其真核表达载体的构建及表达却少见报道。 目的：实验拟构建携带低氧诱导因子1αmu(hypoxia inducible factor 1 alpha,HIF-1α)目的蛋白和人源化海肾绿色荧光蛋白(human renilla reniformis green fluorescent protein,hrGFP)的双基因真核表达载体,并将其转染HEK293A细胞,观测其在细胞中的表达。 方法：利用PCR技术定点突变目的基因供体质粒pCMV6-XL5-HIF1α携带的人HIF1α基因编码区的第402位、564位和803位氨基酸以及去掉其终止密码子,之后在基因序列前后添加新的酶切位点Not Ⅰ和Pvu Ⅰ,酶切、测序检测突变情况,将正确突变的HIF1αmu定向连入腺病毒穿梭载体pShuttle-CMV-IRES-hrGFP-1中。经测序鉴定、Pme Ⅰ酶切线性化后转化BJ5183-AD-1电感受态细胞,通过hrGFP基因荧光表达检测转染情况。 结果与结论：经基因测序证实,HIF-1α基因编码区的第402位、564位和803位氨基酸均定点突变成丙氨酸,终止密码子成功去除。经酶切鉴定及测序证实,重组腺病毒表达载体构建成功。荧光显微镜下观察表明,感染重组腺病毒的HEK293A细胞内有大量绿色荧光表达证实通过Lipofectamine 2000途径可使得重组腺病毒载体成功转染HEK293A细胞。 关键词：低氧诱导因子1α;基因突变;腺病毒载体;绿色荧光蛋白;HEK293A细胞 doi:10.3969/j.issn.1673-8225.2010.46.011     

探讨一种新型Wnt替代蛋白激活脑血管Wnt信号通路对血脑屏障的保护作用

目的探讨一种新型Wnt替代蛋白(Wnt-S)对人脑血管内皮细胞中Wnt/β-catenin信号通路的作用,可能提供一种保护血脑屏障的新途径。方法用分子克隆技术构建Wnt-S in pcDNA3. 1(+)质粒,在HEK293F细胞中瞬时转染该质粒,表达纯化Wnt-S蛋白; Western blot检测含5x His-tag的Wnt-S蛋白;免疫荧光染色检测人源脑血管内皮细胞(hCMEC/D3)标记物CD31、VE-cadherin、GLUT1和MFSD2A;实时荧光定量PCR检测HEK293和hCMEC/D3细胞的Wnt蛋白受体FZD1-10和LRP5/6的表达情况,检测加药处理后HEK293和hCMEC/D3细胞中Wnt/β-catenin信号通路下游靶基因AXIN2的表达情况。结果核酸电泳结果显示Wnt-S质粒构建完成; Western blot结果显示检测到了Wnt-S蛋白; HEK293中FZD1-7和LRP6高表达,hCMEC/D3中FZD2,4,6和LRP6高表达,FZD1,3低表达;实时荧光定量PCR结果显示,加Wnt-S蛋白处理后,HEK293和hCMEC/D3细胞中AXIN2基因表达水平显著上调(P 0. 001),说明Wnt-S蛋白有生物活性,并且激活了Wnt/β-catenin信号通路。结论 Wnt-S蛋白具有与天然Wnt3a蛋白类似的生物功能,能够激活人脑血管内皮细胞中的经典Wnt信号通路,以保护血脑屏障。     

骨髓间充质干细胞中突变型人低氧诱导因子1α真核表达载体的表达

背景：转基因小鼠体内实验证实,重组的低氧诱导因子1α可以促进皮肤形态功能正常的血管新生。 目的：构建能够在常氧条件下同时表达突变型低氧诱导因子1α目的蛋白和绿色荧光蛋白报告分子的新型腺病毒真核表达载体,并转染SD大鼠骨髓间充质干细胞,检测该基因在细胞中的表达情况。 方法：利用Lipofectamine 2000介导将构建成功的重组腺病毒真核表达载体pAd-HIF1αmu- IRES-hrGFP-1转染HEK293A细胞,包装病毒,以最佳感染指数=50将重组腺病毒转染大鼠骨髓间充质干细胞,并设3个对照组,即阳性对照组：转染Ad-CMV-HIF1α-IRES-hrGFP-1;阴性对照组：转染Ad-CMV-IRES-hrGFP-1组;空白组：未转染病毒。 结果与结论：①腺病毒载体成功转染HEK293A细胞,包装成功,细胞内有大量绿色荧光表达。②转染突变型低氧诱导因子1α腺病毒表达载体的细胞在常氧条件下蛋白表达量明显高于转其他3组,3个对照组间差异无显著性意义(P > 0.05)。提示突变型腺病毒真核表达载体Ad-HIF1α-IRES-hrGFP-1在HEK293A内成功包装;突变后低氧诱导因子1α基因能够在常氧条件下大量且高效表达。     

Postnatal development of NR1, NR2A and NR2B immunoreactivity in the visual cortex of the rat

N-Methyl-D-aspartate receptors (NMDARs) are critically involved in some types of synaptic plasticity. The NMDAR subunits NR1, NR2A and NR2B are developmentally regulated, and it has been proposed that developmental changes in their expression may underlie developmental changes in cortical plasticity. Age-dependent change in cortical plasticity is most commonly measured by the monocular deprivation effect, which occurs during a critical period between P22 and P50 in the rat. Although the development of NMDAR subunits has been studied from birth through the fourth postnatal week, there is only meager information from older ages when visual plasticity ends. We hypothesized that there will be significant age-dependent change in expression of NR1, NR2A or NR2B between P22, when the cortex is plastic, and P90, when it is not. We applied specific antibodies recognizing NR1, NR2A and NR2B to the primary visual cortex at P14, P22, P30, P45 and P90. We found age-dependent changes in NR1-IR that were negatively correlated with changes in NR2A-IR; these subunits are not regulated in unison. In contrast, NR2A-IR and NR2B-IR were positively correlated. NR2A-IR and NR2B-IR both passed through a developmental minimum around P45, then recovered to approximately their P22 level. NR1-IR passed through a maximum at P45. There were no significant differences between P22 and P90. These results do not support the simple hypothesis that the loss of plasticity corresponds to a simple transition from juvenile levels of NMDAR subunit proteins to new adult levels. On the other hand, the results do confirm the hypothesis that there are significant changes in processing of NMDAR proteins during the time that plasticity is lost. How these changes of IR relate to synaptic transmission and plasticity needs to be clarified.     

Expression of functional NR1/NR2B-type NMDA receptors in neuronally differentiated SK-N-SH human cell line

The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.     

Distribution and expression of the subunits of N-methyl-d-aspartate (NMDA) receptors; NR1, NR2A and NR2B in hypoxic newborn piglet brains

The purpose of this study was to identify the distribution and the expression of the NR1, NR2A and NR2B subunits of the NMDA receptor after cerebral hypoxia. Ten piglets were divided into control and hypoxic groups (n=5, each). The control piglets were ventilated with normoxia for 1 h, and the hypoxic piglets were ventilated with hypoxia until p_aO₂ was below 20 mmHg. Tissue samples from the nine different regions of newborn piglet brain were obtained, and the protein amount of the NR1, NR2A, and NR2B subunits measured by immunoblot using the antibody to the NR1, NR2A, and NR2B subunits. The NR1, N2A, and NR2B subunits were distributed very differently; hippocampus and cortical area are more prominent than white matter and cerebellum. But the expression of the NR1, NR2A and NR2B subunits were not significantly different between the control and the hypoxic group, 1 h after hypoxic exposure, indicating no changes in the protein amount of NMDA receptor subunits. These results show a significantly higher amount of the NR1, NR2A and NR2B subunits in the hippocampus and the cerebral cortex of newborn brains, indicating that these structures could be highly vulnerable to excitotoxicity in the newborn brain.     

Increased numbers of coassembled PSD-95 to NMDA-receptor subunits NR2B and NR1 in human epileptic cortical dysplasia

PURPOSE: Glutamatergic transmission between neurons occurs at chemical synapses. The N-methyl-d-aspartate (NMDA)-receptor subclass of ionotropic glutamate receptors has been implicated in the epileptogenic mechanisms in human cortical dysplasia (CD). NMDA receptors are clustered at the postsynaptic membrane by anchoring to the postsynaptic density protein PSD-95, a putative ion channel-clustering protein. In this study, we quantitatively investigated the coassembly of PSD-95 to NR2B and NR1 in human epileptogenic cortex as compared with nonepileptic cortex. METHODS: We used coimmunoprecipitation and immunoblotting techniques to quantify and compare the numbers of coassembled PSD-95 with NR2B, PSD-95 with NR1, and NR2B with NR1 in the membrane proteins of brain tissues resected from four patients (aged 3.5, 6, 14, and 18 years) with medically intractable neocortical epilepsy associated with CD. The resected cortical tissues were grouped into epileptic and nonepileptic, as determined by prolonged subdural electrode recordings in three patients and direct intraoperative electrocorticographic recording in one patient. RESULTS: In all patients, the amounts of immunoprecipitated complexes, which reflect the numbers of coassembled PSD-95 proteins to NR2B subunits, were increased in epileptic cortex as compared with nonepileptic cortex. CONCLUSIONS: These results suggest that increased coassembly of NR2B and NR1 with PSD-95 may underlie one of the cellular mechanisms that contribute to the in situ increased hyperexcitability, leading to seizure generation in focal CD.     

盐酸多奈哌齐对拟VaD大鼠海马CA1区NR1及NR2B免疫组织化学表达的影响

目的探讨盐酸多奈哌齐对拟血管性痴呆大鼠海马神经元N-甲基-D-天门冬氨酸(NMDA)受体亚单位R1(NMDAR1,NR1)和R2B(NMDAR2B,NR2B)的免疫组织化学表达的影响。方法采用双侧颈总动脉反复夹闭、再通,并腹腔注射硝普钠法制备模型,用盐酸多奈哌齐溶液灌胃,Y-型迷宫试验观察其行为学改变,用免疫组织化学技术观测大鼠海马神经元NR1、NR2B的表达变化。结果盐酸多奈哌齐组大鼠海马CA1区NR1表达明显降低,与模型组比较有显著性差异(均P<0.01),与假手术组相比差异无显著性;NR2B表达较模型组明显增高(P<0.01),与假手术组比较无显著性差异。结论盐酸多奈哌齐有可能通过降低NR1的表达,提高海马NR2B的表达,从而改善拟血管性痴呆大鼠的学习、记忆成绩。     

Possible protection by notoginsenoside R1 against glutamate neurotoxicity mediated by N‐methyl‐D‐aspartate receptors composed of an NR1/NR2B subunit assembly

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. Wefound that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 μM Glu for 1 hr in a dose‐dependent manner at concentrations from 0.1 to 10 μM, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca²⁺ ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl‐2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N‐methyl‐D ‐aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 μM NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 μM NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain. © 2009 Wiley‐Liss, Inc.     

新生期惊厥对大鼠海马NR1和GABAARα1表达的长期影响

目的 研究新生期惊厥对大鼠海马N-甲基-D-天门冬氨酸(NMDA)受体1(NR1)和γ-氨基丁酸A受体α1亚单位(GABAARα1)表达的长期影响,以期揭示发育期惊厥导致成年大鼠惊厥阈降低的机制.方法 生后6 d的Wistar大鼠48只采用完全随机法分成三氟乙醚吸入惊厥组和对照组,每组各24只,惊厥组再细分为单次惊厥组(诱导惊厥一次,持续30 min)和反复惊厥组(每天诱导惊厥一次,持续30 min,连续6 d),对照组同样操作但不吸入三氟乙醚.分别于惊厥后第7天和第75天取大鼠海马,匀浆提取膜蛋白,应用免疫印记法测定NR1、GABAARα1蛋白表达.结果 单次惊厥组和反复惊厥组75 d的海马NR1表达无显著变化,而反复惊厥组7 d的海马NR1表达较对照组显著增加(P＜0.05).同时,与对照组相比,GABAARα1亚单位在单次惊厥组第75天以及反复惊厥组的表达均有统计学意义(P＜0.05).结论 新生期大鼠反复或单次长程惊厥持续状态能够对海马NR1和GABAARα1表达产生远期影响,这种改变可能在发育期惊厥导致的脑兴奋性提高和惊厥阈降低中起重要作用.     

大鼠生后发育中听皮层NR1 mRNA的表达

目的：研究大鼠生后发育过程中，听皮层神经元NMDA受体NR1 mRNA的表达。方法：采用特异性DIG标记的寡核苷酸探针，分别在动物出生后第7，14，2l，28，35，42，49，56天和成年，检测听皮层NR1 mRNA阳性神经元的分布。结果：NR1亚单位mRNA阳性神经元从出生后第7天即可检测到，之后，随着天龄增加，阳性神经元分布密度呈明显递增趋势。期间，从生后第7天到第14天阳性神经元密度增加最快，增加了33．80％。到生后第35天NR1 mRNA表达达到高峰，一直保持到成年。对称的两侧听皮层阳性神经元表达模式完全一致。结论：听皮层NR1 mRNA表达与动物年龄相关。研究结果为进一步在皮层水平上探讨生后听觉功能发育可塑性的分子机制提供了重要资料。     

Long-term potentiation in the nucleus accumbens requires both NR2A- and NR2B-containing N-methyl-D-aspartate receptors

N-methyl-d-aspartate (NMDA) receptors play crucial roles in several forms of long-term changes in the efficacy of glutamatergic synaptic transmission. The suggestion that the NR2A subunit of the NMDA receptor may be selectively involved in the induction of long-term potentiation (LTP) in the hippocampus and cortex has been challenged. However, the contribution of NR2B in the induction of LTP is not always clearly established. The present study investigates the role of NR2A and NR2B in the induction of LTP in the nucleus accumbens (NAc), a brain region that expresses high levels of NR2B and an NMDA-dependent form of LTP. We recorded extracellular field excitatory postsynaptic potentials/population spikes in slices of mouse NAc. High-frequency stimulation of glutamatergic fibers consistently induced LTP of the field excitatory postsynaptic potential/population spike in the NAc. LTP was abolished in the presence of selective antagonists of either NR2B [R-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol and Ifenprodil] or NR2A ([(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid) subunits. Recordings performed in a low concentration of Mg(2+) ions in the perfusion solution did not reveal a selective involvement of a particular NMDA receptor subunit because either NR2A or NR2B antagonists were able to block LTP. LTP was also abolished in the presence of a low concentration of the non-subunit-selective NMDA receptor antagonist dl-2-amino-5-phosphonopentanoic acid in normal Mg(2+) and low Mg(2+) in the perfusion solution. These results show that the degree of NMDA receptor activation, and not their subunit composition, determines whether LTP is induced in the NAc.