右旋葡萄糖对新生大鼠海马神经元AMPA受体亚基GluR2/3表达的影响

目的:观察右旋葡萄糖对新生大鼠海马神经元细胞谷氨酸离子型受体GluR2/3 mRNA表达的影响。方法:建立原代新生大鼠海马神经元细胞培养方法,培养至第9 d时,给予不同浓度右旋葡萄糖刺激。通过免疫荧光检测海马神经元细胞GluR2表达,通过RT-PCR检测GluR2、GluR3、GluR4 mRNA表达变化。结果:新生大鼠海马神经元细胞培养至第9 d时,可见GluR2呈阳性表达,右旋葡萄糖刺激后海马神经元细胞GluR2mRNA、GluR4mRNA表达明显下调(P0.05);GluR3 mRNA表达明显上调(P0.01)。结论:右旋葡萄糖刺激引起海马神经元细胞GluR受体下调,表现对海马神经元的损伤性影响,可能与糖尿病脑病的发生机理有关。     

P>马桑内酯激活的星形胶质细胞条件培养液及CNQX对正常大鼠脑内及培养的神经元内NSF的影响/P>

目的 揭示星形胶质细胞对神经元内N-乙基顺丁烯二酰亚胺敏感性的融合蛋白(NSF)和AMPA受体的影响及其在癫痫发病中的作用.方法 将马桑内酯激活的星形胶质细胞条件培养液(ACM)注入正常SD大鼠侧脑室,观察其行为变化;运用免疫组织化学方法观察其大脑皮质、海马内NSF免疫反应的变化;将培养的神经元随机分为对照组、ACM组及CNQX ACM组,免疫细胞化学方法观察NSF表达的变化,Western blotting法检测NSF含量的变化.结果 ACM组大鼠在注射后30min出现癫痫行为.免疫组织化学染色结果显示:1.ACM作用后2h及4h,大鼠大脑皮质及海马内NSF免疫反应阳性神经元数和平均吸光度明显降低(P<0.05);2.培养的神经元的免疫细胞化学染色结果显示,ACM组在作用4h及8h时,NSF免疫阳性反应产物与对照组比较明显减少(P<0.05).Western blotting法检测NSF含量,在ACM作用4h及8h时较对照组及CNQX ACM组明显减少(P<0.05).结论 ACM可下调神经元内NSF的表达,并导致动物痫性发作,而运用AMPA受体拮抗剂CNQX则能阻断其下调作用,提示ACM对NSF的下调作用可能与AMPA受体有关.     

激活的星形胶质细胞分泌的TNF-α对大鼠离子型谷氨酸受体1表达的影响

目的探讨星形胶质细胞在癫痫发病中的作用。方法选用肿瘤坏死因子TNF-α(TNF-α)刺激及TNF-α反义寡核苷酸阻断后马桑内酯(CL)刺激纯化培养的海马星形胶质细胞，将这两种条件培养基提取液(ACM)10μl分别注入正常大鼠侧脑室，观察动物行为与脑电图的变化；用免疫细胞化学方法检测大脑皮质与海马中离子型谷氨酸受体(NMDARI)表达水平的改变，并做显微图像分析。结果1．侧脑室注射肿瘤坏死因子TNF-α刺激后的条件培养基提取液可引起大鼠Ⅲ级癫痫样发作及典型的尖波、棘波、棘-慢波癫痫样脑电图表现，大脑前梨状皮质和海马CA1区NMDAR1免疫反应阳性神经元数和平均光密度值均明显高于对照组；2．侧脑室注射TNF-α反义寡核苷酸阻断后由马桑内酯刺激的条件培养基提取液，大鼠无癫痫样行为发生，大脑前梨状皮质和海马CA1区NMDAR1免疫反应阳性神经元数和平均光密度值与对照组无显著性差异。结论1．激活的星形胶质细胞分泌的TNF-α可诱导大鼠癫痫发作。2．NMDAR1表达的变化可能与癫痫发作有关。     

快速老化小鼠P8海马神经元突触可塑性相关的AMPA受体表达异常

目的比较快速老化小鼠P8(SAMP8)与抗快速老化小鼠R1(SAMR1)海马神经元突触可塑性相关的谷氨酸α-氨基-3-羟基-5-甲基-4-异唑丙酸(AMPA)受体表达差异,为阿尔兹海默病(AD)的发病机制提供实验依据。方法取雄性10月龄SAMP8 10只和SAMR1 9只,应用Morris水迷宫实验评价动物学习记忆能力,透射电子显微镜观察海马CA1区神经元突触界面超微结构,蛋白质免疫印迹法检测海马AMPA受体亚基GluR1、GluR2的表达。结果与SAMR1比较,SAMP8逃避潜伏期延长,目标象限时间百分比下降,穿台次数减少;海马CA1区神经元突触后致密带变薄,突触间隙增宽,突触界面曲率下降;海马GluR2含量下降,GluR1含量有下降趋势,但差异无统计学意义。结论海马AMPA受体异常可能是导致突触可塑性受损,引发SAMP8认知障碍的原因之一,AMPA受体在AD的发病中可能占有重要地位。     

精氨酸加压素对大鼠下丘脑视前区AMPA受体GluR1-3蛋白表达的影响

目的研究精氨酸加压素(AVP)对大鼠下丘脑视前区(POA)α-氨基-3-羟基-5-甲基异恶唑-4-丙酸(AMPA)受体GluR1-3 mRNA和蛋白质表达的影响。方法腹腔注射AVP(10μg/kg)或AVP V1a受体抑制剂(30μg/kg)0.5 h后,用反转录实时荧光定量PCR和Western blot检测视前区AMPA受体亚基GluR1、GluR2和GluR3 mRNA和蛋白表达。结果与对照组相比,V1a受体抑制剂明显降低了GluR2 mRNA表达(P0.05);AVP能显著上调视前区AMPA受体亚基GluR1和GluR2蛋白水平(P0.01),减少GluR3蛋白表达(P0.01),但V1a受体抑制剂不能阻断这一作用。结论外源性AVP主要通过影响AMPA受体亚基蛋白表达来调制大鼠视前区AMPA受体介导的谷氨酸能突触传递。     

低氧预适应上调大鼠海马神经元和星形胶质细胞在急性缺氧时的葡萄糖转运蛋白的活性和基因表达

目的 研究低氧预适应对海马神经元和星形胶质细胞在急性缺氧暴露时的葡萄糖转运蛋白的活性和基因表达的影响。方法 培养大鼠海马神经元和星形胶质细胞,每天间歇暴露于低氧混合气体（1% O2、10% CO2、89% N2）20min,连续6d。最后1次低氧暴露24h后,将细胞暴露于无氧混合气体（10% CO2、90% N2）6h,然后立即检测[3H]-2-脱氧葡萄糖（2-DG）的摄取率、GLUT1和GLUT3的mRNA水平及神经元的存活率。结果 低氧预适应上调急性缺氧时神经元和星形胶质细胞的2-DG吸收率、星形胶质细胞GLUT1 mRNA的表达及神经元GLUT1和GLUT3 mRNA的表达,提高神经元存活率,这一作用可被细胞松弛素B消除。结论 低氧预适应上调海马神经元和星形胶质细胞在急性缺氧时的葡萄糖摄取率和葡萄糖转运蛋白的基因表达。     

N-甲基-D-门冬氨酸受体与使君子酸受体在培养海马神经元树突上的膜表面表达与共定位

目的　研究含NR2B亚单位的N 甲基 D 门冬氨酸 (NMDA)受体 (NR2B NMDAR)与含GluR1亚单位的使君子酸 (AMPA)受体 (GluR1 AMPAR)在发育不同阶段的培养海马神经元树突上的膜表面表达与共定位。　方法　以FLAG NR2B和GFP GluR1表达载体共转染原代培养第 5d(DIV5 )的大鼠海马神经元 ,分别用相应特异性抗体和荧光标记二抗作活细胞染色 ,显示膜表面表达的受体簇。　结果　对DIV7和DIV14的培养海马神经元树突表面受体簇计数 (个数 10 0 μm) ,GluR1 AMPAR受体簇分别为 5 9 2± 5 6和 74 8± 3 1(P <0 0 5 ) ;NR2B NMDAR为 38 7±3 5和 80 8± 4 9(P <0 0 1) ;两者共定位为 16 3± 5 2和 4 0 1± 6 0 (P <0 0 1)。DIV14海马神经元 4 0 %的共定位受体簇分布在树突棘上。　结论　随着海马神经元的发育 ,树突膜表面NR2B NMDAR、GluR1 AMPAR及两者共定位受体簇密度均增加 ,提示 ,形成更多具有活性的兴奋性突触 ,且主要位于树突棘上。     

TNFα刺激的星形胶质细胞条件培养液对海马神经元的兴奋作用(英文)

为了探讨肿瘤坏死因子-α(tumor necrosis factor-α,TNFα)刺激的星形胶质细胞对神经元的作用,本研究将体外纯化培养的大鼠海马星形胶质细胞,用反相高效液相法测定TNFα刺激后细胞培养液内谷氨酸的含量;将TNFα刺激后的星形胶质细胞条件培养液(astrocytic conditioned medium,ACM)作用于培养的海马神经元,运用免疫细胞化学方法和图像分析技术研究神经元NF-κBp65和谷氨酸的表达。结果表明:(1)TNFα可明显促进星形胶质细胞释放谷氨酸,(2)ACM作用1 5 min即可诱导神经元NF-κBp65的核表达,30 min达高峰,180 min恢复至对照水平,(3)ACM作用60 min可使谷氨酸免疫反应阳性神经元平均光密度明显升高,持续至240 min。提示,TNFα刺激的星形胶质细胞可通过释放谷氨酸等可溶性物质使神经元快速激活、兴奋性升高。     

TNFα刺激的星形胶质细胞条件培养液对海马神经元的兴奋作用

目的:探讨肿瘤坏死因子-α刺激的星形胶质细胞对神经元的作用。材料和方法:体外纯化培养大鼠海马星形胶质细胞,采用反相高效液相法测定TNFα刺激后细胞培养液内谷氨酸的含量;将TNFα刺激后的星形胶质细胞条件培养液（Astrocytic Conditioned Medium,ACM）作用于培养的海马神经元,运用免疫细胞化学方法和图像分析技术研究神经元NF-κBp65和谷氨酸的表达。结果:（1）TNFα可明显促进星形胶质细胞释放谷氨酸;（2）ACM作用15min即可诱导神经元NF-κBp65的核表达,30min达高峰,180min恢复至对照水平;（3）ACM作用60min可使谷氨酸免疫反应阳性神经元平均光密度明显升高,持续至240min。结论:TNFα刺激的星形胶质细胞可通过释放谷氨酸等可溶性物质使神经元快速激活、兴奋性升高。     

S100A9通过增加小鼠星形胶质细胞的谷氨酸分泌促进神经元损伤

目的:研究钙防卫蛋白S100A9刺激体外星形胶质细胞后细胞外谷氨酸浓度变化及其对神经元影响和调控机制。方法:分离培养新生C57BL/6小鼠大脑皮层星形胶质细胞和神经元；Amplite荧光法检测星形胶质细胞培养上清谷氨酸浓度；Real time RT-PCR和Western Blot分别检测星形胶质细胞谷氨酸转运体(GLT-1) mRNA和蛋白表达；Fluo-4荧光探针法检测星形胶质细胞胞内Ca²⁺浓度；转录组测序并结合KEGG分析筛选星形胶质细胞谷氨酸浓度变化的调控机制；S100A9刺激星形胶质细胞的培养上清(CS)干预神经元后，末端脱氧核苷酸转移酶标记法(TUNEL)检测神经元凋亡，CCK-8试剂盒检测神经元存活。结果:S100A9刺激星形胶质细胞后细胞外谷氨酸浓度增加，GLT-1 mRNA和蛋白表达减少，细胞内Ca²⁺浓度增加。差异表达基因KEGG富集在核因子-κB(NF-κB)信号通路、toll样受体(TLRs)信号通路和肿瘤坏死因子(TNF)信号通路等。培养上清组神经元凋亡率高于对照组。结论:S100A9可能通过激活TLR4/NF-κ...     

Aging-related alterations in the expression and distribution of GluR2 and PICK1 in the rat hippocampus

Deficit in synaptic plasticity in the hippocampus frequently occurs during normal aging. Although the protein level and calcium permeability of AMPARs alter with aging, the alteration of AMPARs and their regulatory proteins during aging are far from understanding. Dynamics of GluR2 subunit are dependent on the function of protein interacting with Cα kinase 1 (PICK1), PKCα and calcineurin (CaN). Here, we firstly show that the expression of PICK1 and CaN B decreased significantly in the hippocampus of old rats compared to that of young and adult rats. The decrease was accompanied by a reduction of GluR2 and PKCα and an increase in CaN A. Next, we found that in young and adult rats, the distribution of PICK1 and GluR2 diffused in the cytoplasm of hippocampal neurons, but closely around perinuclear in the hippocampal neurons of old rats. These results suggest that the expression of GluR2, PICK1, PKCα and CaN B significant decreased in the hippocampus and these alterations may lead to altered distribution of GluR2 and PICK1 during aging.     

Subunit interaction with PICK and GRIP controls Ca2+ permeability of AMPARs at cerebellar synapses

At many excitatory central synapses, activity produces a lasting change in the synaptic response by modifying postsynaptic AMPA receptors (AMPARs). Although much is known about proteins involved in the trafficking of Ca2+-impermeable (GluR2-containing) AMPARs, little is known about protein partners that regulate subunit trafficking and plasticity of Ca2+-permeable (GluR2-lacking) AMPARs. At cerebellar parallel fiber-stellate cell synapses, activity triggers a novel type of plasticity: Ca2+ influx through GluR2-lacking synaptic AMPARs drives incorporation of GluR2-containing AMPARs, generating rapid, lasting changes in excitatory postsynaptic current properties. Here we examine how glutamate receptor interacting protein (GRIP, also known as AMPAR binding protein or ABP) and protein interacting with C-kinase-1 (PICK) regulate subunit trafficking and plasticity. We find that repetitive synaptic activity triggers loss of synaptic GluR2-lacking AMPARs by selectively disrupting their interaction with GRIP and that PICK drives activity-dependent delivery of GluR2-containing receptors. This dynamic regulation of AMPARs provides a feedback mechanism for controlling Ca2+ permeability of synaptic receptors.     

激活的星形胶质细胞条件培养液对神经元内iNOS、CaMKⅡ及AC表达的影响

为探讨星形胶质细胞(Ast)在癫痫发病机制中的作用,本研究通过体外分离纯化培养分别获取大鼠大脑皮质Ast及神经元,用睫状神经营养因子(CNTF)激活Ast,用其培养液,即星形胶质细胞条件培养液(ACM)孵育神经元4、8、12h后,采用Westernblot法及图像分析技术观察ACM对神经元内表达钙调蛋白依赖性激酶II(CaMKⅡ)、诱导型一氧化氮合酶(iNOS)及腺苷酸环化酶(AC)的影响。结果表明,ACM作用神经元4h即可引起CaMKⅡ、iNOS及AC的表达增多并持续至12h(P<0.05),iNOS在8h最为明显,AC的表达在12h最为明显。上述结果提示ACM中含有致痫因子,NOS-NO-cGMP,Ca2+/CaM-CaMKⅡ及AC-CaMP-PKA三条信号通路可能参与其致痫机制中的信号转导。     

Effect of antibodies against AMPA glutamate receptors on brain neurons in primary cultures of the cerebellum and hippocampus

Rabbit antibodies against GluR₁ subunit of AMPA glutamate receptors in a concentration of 1 μg/ml significantly increased intracellular Ca²⁺ concentration and decreased mitochondrial potential in hippocampal neurons, i.e. produced changes typical of the influence of glutamate in toxic concentrations. In cerebellar neurons rabbit antibodies potentiated glutamate-induced increase in intracellular Ca²⁺ concentration and significantly decreased the mitochondrial potential (compared to the level observed after application of glutamate alone). The exposure of cultured cerebellar neurons to antibodies in a concentration of 0.1 μg/ml for 24 h was followed by a 50% decrease in ATP concentration and development of neuronal necrosis. Our results attest to an important role of autoimmune damage to neurons during hyperstimulation of glutamate receptors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 59–62, July, 2006     

Upregulation of GluR2 decreases intracellular Ca2+ following ischemia in developing gerbils

Developing animals are known to be resistant to cerebral ischemia. To investigate the mechanisms by which developing animals exhibit ischemic resistance, we examined the changes in intracellular calcium ([Ca2+]i) after oxygen-glucose deprivation (OGD) using hippocampal slices from gerbils. We found that increases of [Ca2+]i in hippocampal CA1 neurons is significantly less after OGD in developing gerbils than in adults. Western blot analysis of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors (AMPARs) showed that GluR2 expression, but not that of the other AMPARs is significantly higher in developing gerbils than in adults. Expression of the anti-apoptotic proteins such as HSP70, Bcl-XL, and plasma membrane Ca2+-ATPase type1 (PMCA1) are not higher in the developing gerbils than in adults. These results suggest that the higher expression of GluR2 is important for the smaller increases in [Ca2+]i and enhanced resistance to ischemia-induced neuronal damage in developing animals.     

Increased expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 within dorsal root ganglia in a rat model of bone cancer pain

Astrocytes were reported to show neuroprotective effects on neurons, but there was no direct evidence for a functional relationship between astrocytes and neural stem cells (NSCs). In this experiments, we examined neuronal differentiation of NSCs induced by protoplasmic and fibrous astrocytes in a co-culture model respectively. Two types of astrocytes and NSCs were isolated from E13 to 15 cortex of rats. The neuronal differentiation of NSCs was examined after co-culture with two kinds of astrocytes. There were more neuronal marker β-tublin III positive cells from NSCs co-cultured with protoplasmic astrocytes. However the differentiated neurons, whether co-cultured with protoplasmic astrocytes or fibrous astrocytes, both expressed glutamate AMPA receptor subunit GluR2 protein and exhibited biological electrical reactivity after stimulated by glutamine. Therefore, these findings indicated that two types of astrocytes could induce the differentiation of NSCs and also possibly induce functional maturation of differentiated neurons, among which protoplasmic astrocytes have the ability to promote neuronal differentiation of NSCs compared with fibrous astrocytes.