新生大鼠海马神经元的体外原代培养

目的研究体外原代培养新生大鼠海马神经元的方法,并观察其发育分化过程中形态学的变化。方法取出生24 h内的Wistar大鼠分离海马,消化后差速贴壁,种植在涂有多聚-L-赖氨酸的盖玻片上,于不同时间在倒置相差显微镜下观察形态变化;采用NSE免疫细胞化学技术鉴定神经元。结果神经元12~24 h后大部分贴壁,随时间延长形态多变,突起逐渐增多,神经元突起间互相接触形成网络。培养第7~10天时细胞最为丰满,至28天时培养液中有悬浮的细胞碎片。NSE鉴定结果显示神经元纯度为(92.3±6.8)%。结论该方法简单易行,神经元纯度较高,可作为神经元体外培养的良好模型用于以后的研究。     

蝎毒耐热蛋白对原代培养海马神经元NPY表达的影响

探讨具有抗癫痫反复发作作用的蝎毒耐热蛋白(scorpion venom heat-resistant protein,SVHRP)对大鼠海马神经元神经肽Y(neuropeptide Y,NPY)表达的影响。采用免疫细胞化学ABC法与HPIAS系列彩色病理图文定量分析系统相结合,检测NPY-免疫阳性产物(NPY-IR)表达的变化,用RT-PCR技术观察NPY mRNA表达水平的变化。终浓度范围为0.2、2、20和200μg/ml的SVHRP分别与培养10d的海马神经元共孵育24h后,NPY阳性神经元的反应强度明显增强,具有剂量-反应关系。终浓度为20μg/ml的SVHRP分别与培养10d的海马神经元共孵育3、6、9、12和24h,NPY阳性反应强度增强,并呈时间-反应关系。RT-PCR结果显示,20μg/ml的SVHRP与原代培养海马神经元共孵育24h后,NPY mRNA的表达明显增加。以上结果提示SVHRP可诱导原代培养海马神经元NPY阳性反应和NPY mRNA的表达。     

胰岛素对糖尿病大鼠海马内神经营养因子-3阳性神经元表达的影响

目的：观察糖尿病大鼠海马中神经营养因子-3(NT-3)的表达变化及胰岛素治疗对其表达的影响。方法：将雄性大鼠随机分为对照组,糖尿病1,3月组,胰岛素治疗1,3月组,STZ 腹腔注射制模,测体重与血糖值并取海马行 HE 和免疫组化 SABC 法染色,计算机图像分析系统测平均光密度值。结果：血糖在糖尿病组比对照组显著升高;胰岛素组与对照组无显著性差异。海马各区 NT-3免疫阳性神经元的数量及阳性强度在糖尿病组有不同程度降低,以 CA1区最明显。胰岛素组则不降低。结论：糖尿病大鼠海马神经元 NT-3表达减弱,胰岛素治疗可使海马神经元 NT-3表达恢复正常。     

四君子汤对阿尔茨海默病大鼠中枢海马炎症因子及NLRP3小体的影响

目的 探讨四君子汤（SiJunZi Decoction, SJZD）治疗大鼠阿尔茨海默病的作用机制。方法 将60只雄性SPF级大鼠随机分为正常组,模型组,双氢麦角碱（dihydroergoline, DHE）组,SJZD低、中、高治疗组。采用Morris水迷宫实验检测大鼠记忆功能的变化;ELISA法检测IL-1β、IL-6及IL-10含量;免疫组化法检测caspase11蛋白表达变化;Western blot法检测海马组织NLRP3、caspase-1、caspase-4、caspase-5蛋白表达。结果 与正常组比较,模型组大鼠潜伏逃避期时间增加（P<0.01）、穿越次数减少（P<0.01）,IL-1β、IL-6含量升高（P<0.01）,IL-10含量降低（P<0.01）,NLRP3、caspase-1、caspase-4、caspase-5、caspase-11(P<0.01)表达上调;DHE与模型组比较,潜伏逃避期减少（P<0.01）,穿越次数增加（P<0.01）,IL-1β减少（P<0.01）,NLRP3、caspase-1表达下...     

新生大鼠离体海马神经元原代培养方法及鉴定

目的:建立一种稳定的生后大鼠离体海马神经元的培养方法。方法:无菌环境下将出生24 h内SD大鼠断头取脑分离海马,经消化后差速贴壁,采用无血清培养基进行培养,倒置显微镜下观察不同时间段细胞生长形态变化:采用Hoechst33258与NeuN抗体双染方法鉴定神经元。结果:采用差速贴壁后,使用无血清培养基培养的神经元生长良好,纯度较高,12 d时经鉴定神经元纯度可以达到92%以上。结论:差速离心法后可以采用无血清培养神经元,培养的神经元具有纯度高,结果稳定的优点,该方法为以后进行相关的研究奠定了基础。     

原代培养海马神经元Drebrin和Synaptophysin的表达与动态变化

目的:观察原代海马神经元不同时期树突棘的变化以及树突棘蛋白Drebrin和突触囊泡膜蛋白SYN的表达水平。方法:光镜观察原代培养7、14和20 d的海马神经元的形态改变。应用免疫荧光对Drebrin和SYN进行染色,观察树突棘形态和数量的改变。进一步应用Western Blot检测Drebrin和SYN蛋白的表达变化。结果:培养7 d的海马神经元,未见树突棘结构,SYN斑点状阳性产物较少。随着海马神经元培养时间的加长,突起数量逐渐增加,14 d和20 d的树突棘数量和SYN阳性产物均显著增加。Drebrin和SYN蛋白表达水平也随培养时间的加长而显著增加。结论:原代培养的海马神经元在14 d已有树突棘形成,20 d显示出成熟树突棘的形态和数量。Drebrin和SYN的蛋白表达水平反映了树突棘和突触的形成和功能状态。     

睫状神经营养因子对应激引起海马CA3神经元损害的作用

目的;探讨睫状神经营养因子对应激引起海马ＣＡ３区神经元损害的作用。方法：采用Ｂｉｅｌｓｃｈｏｗｓｋｙ－Ｇｒｏｓ－Ｌａｗｒｅｎｔｊｅｗ染色法和常规透射电镜技术,观察急性和慢性足底电击大鼠的海马ＣＡ３区神经元开矿的变化,及双侧海马注射睫状神经营养因子对其影响。结果;急性应激大鼠海马神经元形态无明显变化;慢性应激大鼠海马神经元出现明显的损伤性形态变化;睫状神经营养因子对正常大鼠和急性应激大鼠的海马神经元     

胎鼠海马神经元原代培养及转染条件的优化

目的 建立一种稳定的大鼠胎鼠海马神经元培养方法并能够有效的鉴定其纯度,探讨用化学转染试剂体外转染大鼠胎鼠海马神经元的最佳转染条件.方法 分离Wista大鼠E18d胚胎并取海马组织,无血清培养基培养并做MAP2荧光染色鉴定其纯度.应用lipofectamine <'TM>2000转染试剂盒,大鼠神经元电转试剂盒,lipo...     

新生大鼠海马神经元原代无血清培养与鉴定

 摘要:目的 建立一种简单、易行的海马神经元无血清体外培养方法以获得高纯度、高活力的海马神经元。方法 取新生24h内SD大鼠,分离海马,经消化后种植于包被有Matrigel基膜的玻片上,24h后换含有N2、B27的neurobasal无血清培养基,于不同时间在倒置相差显微镜下观察细胞的生长状态;采用β-tublinⅢ免疫荧光细胞化学技术鉴定海马神经元纯度。结果 大部分海马神经元于3-24h贴壁且可长出细长突起,3d细胞具有典型神经元的形态特征, 5d神经突起进一步增多并形成稠密的网络连接,7d神经元胞体丰满,趋于成熟。经β-tublinШ免疫荧光技术鉴定纯度为（94.2±3.6）%。结论 此方法简单有效,可获得高纯度的海马神经元。     

线粒体缺陷对原代培养新生大鼠海马神经元微管相关蛋白表达的影响

目的：观察线粒体呼吸链复合体IV（即细胞色素C氧化酶）抑制剂叠氮钠（NaN₃）对神经元细胞骨架系统的影响,探讨线粒体缺陷在阿尔茨海默病发病中的作用。方法：将叠氮钠与原代培养的新生大鼠海马神经元共同孵育,MTT法测定细胞存活率,激光共聚焦显微镜系统观察细胞微管结构及微管相关蛋白表达的变化。结果：叠氮钠8-128 mmol/L与培养的神经元共孵育6-24 h,细胞存活率下降,呈时间及剂量依赖性;NaN₃ 16、64 mmol/L作用6 h,使细胞微管结构损伤,微管相关蛋白表达下降,尤以突起内最为显著。结论：叠氮钠与培养的神经元共孵育导致神经元损伤,其机制与线粒体缺陷致细胞骨架系统异常有关。     

磁刺激对小鼠海马原代神经元即刻早期基因和细胞骨架蛋白表达的影响

目的:观察磁刺激对小鼠海马原代神经元c-fos、活性调节的细胞骨架蛋白(Arc)和微管相关蛋白2(MAP2)的表达及神经元突起生长的影响,探讨磁刺激对细胞突起生长的影响机制.方法:体外培养的海马原代神经元,分为对照组,假刺激组,20％(20％％强度组)、30％(30％强度组)、40％最大刺激强度组(40％强度组),24 h后开始刺激,频率1 Hz,最大输出强度3.7T.连续刺激5d后应用荧光显色检测c-fos、Arc和MAP2阳性神经元免疫荧光强度,计数MAP2阳性神经元多突起神经元个数及细胞突起长度,并应用免疫印迹和RT-PCR技术对免疫荧光显色结果进行验证.结果:磁刺激能促进小鼠海马原代神经元突起数目和长度的生长,3个强度组多突起神经元(n≥2)占总神经元的比例以及海马原代神经元突起长度均高于对照组,且30％强度组高于20％、40％强度组,结果有统计学意义.免疫荧光显色30％强度组c-fos阳性神经元比例、Arc和MAP2免疫荧光强度高于相关对照组,差异有统计学意义.免疫印迹和RT-PCR结果同免疫荧光显色结果一致.结论:磁刺激能促进体外培养的海马原代神经元突起生长,其机制可能和c-fos、Arc和MAP2的表达上调有关.     

Alternative modalities of adenovirus-mediated gene expression in hippocampal neurons cultured on microisland substrate

Previously, we have used CsCl gradient-purified recombinant adenovirus (AdV) to successfully transfer genes into hippocampal neurons cultured on microisland substrate. Here, we report that purification of AdV particles is not required and efficient gene expression can be achieved using either crude AdV lysates or HEK 293 cells infected with AdV. The advantages of the simplified procedure are greatly reduced preparation time and reduced requirements for equipment and expertise.     

Silencing-induced metaplasticity in hippocampal cultured neurons

Silencing-induced homeostatic plasticity is usually expressed as a change in the amplitude or the frequency of miniature postsynaptic currents. Here we report that, prolonged (approximately 24 h) silencing of mature (20-22 days in vitro) cultured hippocampal neurons using the voltage-gated sodium channel blocker tetrodotoxin (TTX) produced no effects on the amplitude or frequency of the miniature excitatory postsynaptic currents (mEPSCs). However, the silencing changed the intrinsic membrane properties of the neurons, resulting in an increased excitability and rate of action potentials firing upon TTX washout. Allowing neurons to recover in TTX-free recording solution for a short period of time after the silencing resulted in potentiation of mEPSC amplitudes. This form of activity-dependent potentiation is different from classical long-term potentiation, as similar potentiation was not seen in nonsilenced neurons treated with bicuculline to raise their spiking activity to the same level displayed by the silenced neurons during TTX washout. Also, the potentiation of mEPSC amplitudes after the recovery period was not affected by the N-methyl-d-aspartate receptor blocker d-2-amino-5-phosponopentanoic acid or by the calcium/calmodulin-dependent kinase II (CaMKII) inhibitor KN-62 but was abolished by the L-type calcium channel blocker nifedipine. We thus conclude that the potentiation of mEPSC amplitudes following brief recovery of spiking activity in chronically silenced neurons represents a novel form of metaplasticity that differs from the conventional models of homeostatic synaptic plasticity.     

Developmental change in ganglioside expression in primary culture of rat neurons

Developmental changes in ganglioside levels and patterns were investigated in neuronal cells dissociated from 17-day-old fetal rat hemispheres for up to 7 days of culture. Increases in ganglioside contents and the onset of GM3 synthesis, which is associated with proliferation of glial cells, were observed as the neuronal network was established in cell cultures. The distribution of gangliosides in developing neurons was monitored by the indirect immunofluorescent technique using three anti-ganglioside antibodies. Anti-GM1 antibody showed immunofluorescence only on the cell soma 1 and 3 days after plating and additional binding between cell aggregates by 7 days in culture. GD3 ganglioside, the predominant species in embryonic neurons, was not detected on the neuronal cell surface, whereas the number of positively stained non-neuronal cells was increased at 7 days. Monoclonal A2B5 antibody suggested that polysialogangliosides play a role in neuronal network formation. In 1-day-old culture, however, all antibodies bound poorly to cell surface antigens and strongly to cells, the membranes of which were permeabilized with acetone. These results suggest that a substantial amount of gangliosides are retained, transformed within the cell to more complex gangliosides, and translocated to the cell surface following neurite outgrowth and morphological changes.     

ATP诱导大鼠原代培养海马神经元神经毒性及对GAP-43表达的影响

目的:观察不同浓度ATP对大鼠原代培养海马神经元的损伤作用及GAP-43表达的影响,并探讨其可能机制。方法:原代培养新生SD大鼠海马神经元至第8 d,MTT法检测不同浓度ATP作用下海马神经元的存活率,倒置相差显微镜观察海马神经元的形态学改变,免疫荧光细胞化学法观察损伤后海马神经元GAP-43的表达变化,钙离子荧光染料Flou-4/AM检测细胞内钙离子的浓度变化。结果:低浓度ATP对海马神经元无明显影响,高浓度ATP对海马神经元有毒性损伤作用,且呈剂量依赖性。P2X受体拮抗剂可阻断ATP介导的细胞毒性作用,但P2X7受体拮抗剂的阻断作用不明显。高浓度ATP作用海马神经元4 h后,GAP-43的表达水平明显上调,且表达部位发生改变,主要表达于胞体,突起表达减少甚至消失。高浓度ATP组荧光衰减速率明显减慢,说明高浓度ATP参与了海马神经元细胞内钙调节。结论:高浓度ATP对海马神经元有毒性损伤作用,损伤后GAP-43代偿性表达增多,且与细胞内钙离子浓度变化有关,P2X7受体可能不是介导ATP此作用的主要受体亚型。     

Molecular correlates of the M-current in cultured rat hippocampal neurons

M-type K⁺ currents ( I _K(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K⁺ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I _K(M) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I _K(M) with an IC₅₀ of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed I _K(M) with an IC₅₀ of 0.7 ± 0.1 m m . In the remaining cells, TEA maximally reduced I _K(M) by only 59.7 ± 5.2 % with an IC₅₀ of 1.4 ± 0.3 m m ; residual I _K(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I _K(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.     

CRMP-2 induces axons in cultured hippocampal neurons

In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant-negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.