Rho激酶影响大鼠海马神经元突起生长的实时成像

目的:观察Rho激酶对大鼠海马神经元突起生长的影响.方法:体外培养新生大鼠海马神经元5 d后,连续动态观察溶血性磷脂酸(lysophosphatidic acid,LPA)及Rho激酶抑制剂Y-27632对神经元突起生长的影响.结果:对照组神经元一级突起迅速生长、延伸,不断发分支,形成丰富的二级、三级突起;用LPA处理后,神经元大部分突起进行性塌陷,一级突起逐渐缩短并且变细,二级、三级突起数量减少,且较细小;而预先用Y-27632处理后再加LPA,细胞突起塌陷的现象减少,神经元的突起不断分支、延伸,一级、二级、三级突起数量较LPA组增多且长度也增加.结论:Rho激酶参与LPA诱导海马神经元突起回缩的过程,抑制Rho激酶的活性可抑制LPA诱导的突起回缩.     

RhoA/Rho激酶在大鼠阴茎勃起机制中的调节作用

一般认为一氧化氮（nitric oxide,NO）释放增加促进小动脉和海绵体平滑肌舒张是导致男性阴茎勃起的主要机制。研究发现内源性血管收缩因子对维持阴茎萎状态有重要作用。应用大鼠模型活体检测使用Y-27632拮抗Rho-激酶活性后对阴茎勃起生理机制的影响;应用免疫转印技术检测 阴茎海绵体内RhoA和Rho激酶蛋白的表达。结果显示在大鼠海绵体组织内有内源性RhoA和Rho激酶蛋白的表达和存在;Y-27632海绵体内注射阻滞RhoA/Rho激酶活性使ICP和CCP/MAP比值明显升高;局部小剂量应用Y-27632对MAP没有明显影响;Y-27632可增强系列电刺激引起的由NO介导的CCP/MAP比值的增加;NO合成酶和鸟苷酸环化酶抑制剂的作用不能阻滞RhoA/Rho激酶抑制剂对大鼠阴茎海绵体平滑肌的松弛作用和CCP/MAP的增加。说明RhoA/Rho激酶信号系统发挥了维持海绵体萎软状态重要作用,这是与NO介导途径不同的阴茎勃起生理调节机制。RhoA/Rho激酶抑制剂可能是ED治疗的新领域新方法。     

大鼠海马发育过程中突触蛋白-Ⅰ和生长相关蛋白-43 mRNA的表达

目的:探讨大鼠海马突触蛋白和生长相关蛋白-43 mRNA的表达,了解神经元的发育。方法:采用RT-PCR方法检测大鼠发育不同阶段突触蛋白和生长相关蛋白-43 mRNA量的变化。结果:RT-PCR产物电泳后在731 bp、984 bp、324 bp处出现清晰的电泳条带,实际扩增长度与设计长度相吻合,未出现与DNA相同的杂带。内参照β-actin的PCR产物电泳条带在孕期、新生和成年时亮度均一。突触蛋白的PCR产物电泳条带以成年大鼠表达最强,胎鼠次之,新生大鼠较弱。生长相关蛋白-43的PCR产物电泳条带以新生鼠亮度最强,胎鼠次之,成年大鼠最暗。结论:大鼠海马在新生期处在轴突生长旺盛的时期,而在胚胎期和成年期是突触形成和建立的主要阶段。     

CRMP2蛋白促进海马神经元树突野的形成

目的:探讨坍塌反应调节蛋白2(CRMP2)对海马神经元树突野形成的作用.方法:培养大鼠海马神经元,用基因转染的方法检测CRMP2蛋白的作用,免疫荧光显示树突,全细胞膜片钳检测微小兴奋性突触后电流(mEPSCs).结果:过表达CRMP2促进树突的生长以及分支形成,而敲减CRMP2抑制树突的生长和形成新的分支,CRMP2基因促进树突野的形成,促使树突野变得复杂(P0.05);全细胞膜片钳检测显示:CRMP2促进形成的树突能够表达AMPA受体,而且可以诱导出mEPSCs,而敲减CRMP2抑制树突AMPA受体,以及降低mEPSC的幅度和频率,其改变有统计学差异(P0.05).结论:CRMP2促进功能性树突形成,并提高树突野的复杂性.     

大鼠海马锥体神经元外向电流特征分析

利用全细胞膜片钳技术研究了大鼠海马锥体神经元去极化激活的外向电流的特征.结果表明,短时去极化至-60mV以上电位可引发快速上升的外向电流,之后缓慢衰减至一平台.当保持电位改变时,该峰电流与平台电流的幅度均会发生变化,但前者较后者变化显著.试验中测得峰电流与平台电流的逆转电位分别为-(76.1±5.97)mV和-(83.6±4.13)mV,与用Nerst方程计算出的本试验细胞外液与电极内液的钾离子平衡电位Ek=-88mV接近,说明该外向电流是钾电流,且提示此外向电流包括两种成分.试验结果还表明,此外向电流的衰减过程可用双指数方程很好地拟合,而在500ms预脉冲刺激后再经去极化激活的快成分的失活过程则适合用单指数方程进行拟合,从而进一步证实大鼠海马锥体神经元的外向钾电流包括快慢两种成分(IA和IK).IA的稳态激活和失活过程均可用Boltzmann方程进行拟合,半数激活和半数失活电压分别为(7.40±3.01)mV和-(66.27±3.62)mV,但其稳态失活在测试电压范围内(-100mV至+30mV)不完全.     

大鼠海马神经元neurobasal无血清的原代培养方法

目的:建立纯度和活力较高的无血清原代培养海马神经元的方法。方法:新生SD大鼠海马,用neuro-basal培养基培养,免疫荧光鉴定神经元纯度,MTT法检测其活力。结果:神经元接种12~24 h后贴壁,并长出细小突起,3 d具有典型神经元形态特征,4 d突起形成稀疏的神经纤维网络,8 d后神经元5~10个聚集成团,突起密集,生长稳定,12 d后出现细胞碎片。Tubulin荧光染色显示清晰的神经元,突起绵长且相互交织,占细胞总数的66.7%;GFAP荧光染色的细胞数量少,突起短粗,占33.7%。培养1~5 d MTT代谢率逐渐上升,6~11 d处于平台期,11 d后下降。结论:neurobasal无血清培养获得的神经元纯度大于60%,6~11 d的细胞适于细胞学实验。     

HO-1蛋白在大鼠海马神经元中的发育

通过免疫组化及流式细胞仪计数等方法,观察生后P0～P300大鼠海马中血红素氧化酶-1蛋白阳性细胞的形态、数量与比例变化,研究血红素氧化酶-1蛋白在大鼠海马中的发育表达．结果表明：血红素氧化酶-1蛋白阳性细胞高峰出现在P14～P21之间,包括海马CA1,CA2 CA3,DG区,P0,P7,P30,P90 P300血红素氧化酶-1蛋白逐渐减弱．结果提示：血红素氧化酶-1在大鼠海马发育过程中的表达,可能受到海马特殊区域中不同类型细胞的调节．     

大鼠海马发育过程中CRMPs家族mRNA的表达

目的:探讨大鼠海马发育过程中坍塌反应调节蛋白(CRMPs)家族mRNA的表达规律.方法:用RT-PCR方法检测大鼠发育不同阶段CRMPs家族mRNA的表达.结果:RT-PCR实际扩增长度与设计长度相吻合.内参照β-actin电泳条带在发育各时期灰度无明显差异.CRMP-1 mRNA以新生鼠和幼年鼠表达最多,成年大鼠弱表达(P<0.05).CRMP-2从胚胎鼠至幼年鼠呈现逐渐增加的趋势,成年鼠相对较低(P<0.05).CRMP-3以幼鼠和成年大鼠表达较多,其中幼鼠表达最多(P<0.05),新生鼠表达最少(P<0.05).CRMP-4在胎鼠、新生鼠和幼年鼠表达量接近,成年鼠表达相对较低(P<0.05).CRMP-5在各个阶段均呈较高表达,以新生鼠和成年鼠表达较多,其中成年大鼠表达最多(P<0.05),胎鼠表达最弱(P<0.05).结论:从胚胎期至幼年期,CRMPs家族除CRMP-4稳定于高表达水平外,其余成员表达量均逐渐增多,其中CRMP-3和CRMP-5区别于其他成员,在成年期表达量继续增高.     

地黄饮子含药脑脊液对海马神经元损伤的影响

探讨地黄饮子对β-淀粉样蛋白(Aβ25-35)诱导体外原代培养海马神元损伤的保护作用．把离体培养的海马神经元分为6组：空白对照组、模型组、、VE组、中药低剂量组、中药中剂量组和中药高剂量组,分别测定细胞培养液中乳酸脱氢酶(LDH)含量和MTT自动比色微量分析,观察中药地黄饮子脑脊液对受损神经元的影响．表明中药地黄饮子脑脊液能明显降低Aβ25-35诱导的海马神经元细胞培养液中LDH含量,提高细胞生存活力．地黄饮子有效成分能穿透血脑屏障,对抗Aβ25-35诱导的海马神经元损伤,表现出对海马神经元一定的保护作用．     

大鼠美解眠点燃效应的形成与海马神经元凋亡

大鼠美解眠点燃效应的形成与海马神经元凋亡刘新峰金泳清陈光辉张隽吴波＊南京大学医学院临床学院南京军区南京总医院神经内科（南京，２１０００２）＊病理科关键词大鼠；美解眠；海马；点燃效应，神经元凋亡分类号Ｒ９７１．７０引言美解眠是一种速效的中枢兴奋剂，可...     

Polarized sorting of glypiated proteins in hippocampal neurons.

Our recent studies suggested that neurons and epithelial cells sort viral glycoproteins in a similar manner. The apical influenza virus haemagglutinin was preferentially delivered to the axon of hippocampal neurons in culture, whereas the basolateral vesicular stomatitis virus glycoprotein was sorted to the dendrites. To investigate whether other membrane proteins showed similar sorting in neurons and epithelial cells, we have analysed the localization of a glypiated (glycosylphosphatidylinositol anchored) protein, Thy-1, in hippocampal neurons in culture. In MDCK and other epithelial cells, endogenous glycosylphosphatidylinositol (GPI)-anchored proteins, as well as mutated exogenous proteins containing the GPI-attachment signal, undergo preferential delivery to the apical surface. This polarized sorting of GPI-anchored proteins has been proposed to occur by the same mechanisms as the sorting of glycolipids to the apical surface. We report here that the neuronal GPI-protein Thy-1 is present in hippocampal neurons in culture and is exclusively located on the axonal surface. This finding further strengthens our hypothesis that the mechanisms of sorting of surface components may be similar in neurons and epithelial cells.     

Effect of ytterbium on sodium current and its kinetics in rat hippocampal neurons

This study addressed the effects of Yb3＋ on voltage-gated sodium currents in rat hippocampal neurons using the whole-cell patch-clamp technique. Voltage-clamp recordings in single neurons were filtered and stored in a computer. Yb3＋ increased the amplitude of sodium currents in a concentration-dependent and voltage-dependent man- ner. The 50 % enhancement concentration of Yb3＋ on sodium currents was about 8.97 μmol/L, which was dif- ferent from the inhibitory effects of Yb3＋ on potassium current. The analysis on the activation and inactivation kinetics of Na＋ current showed that 100 μmol/L Yb3＋ did not change the process of activation and inactivation. In addition, the times reaching the peak of current （t） and inactivated time constant （τ） were voltage dependent. 100 μmol/L Yb3＋ significantly prolonged the time to peak at -70 and -80 mV. The effect disappeared at the positive direction of -70 mV. Furthermore, Yb3＋ decreased r val- ues to more positive values than -80 mV. In total, Yb3＋ did not change the process of activation, but impelled inacti- vated process. Yb3＋ mainly increased the Na＋ current through changing its conductance. It might be one of the mechanisms that Yb3＋ affected the hippocampal neurons.     

Achyranthes bidentata Blume extract promotes neuronal growth in cultured embryonic rat hippocampal neurons

We have prepared an aqueous extract of Achyranthes bidentata Blume, a commonly prescribed Chinese medicinal herb, and reported, in previous studies, that A. bidentata extract benefits nerve growth and prevents neuron apoptosis. In this study, we investigated the actions of A. bidentata extract on survival and growth of primarily cultured rat hippocampal neurons. The morphological observation revealed that neurite growth from hippocampal neurons was significantly enhanced by A. bidentata extract with similar effects to those induced by nerve growth factor (NGF), and the greatest neurite growth appeared on treatment with A. bidentata extract at 1 μg/ml for 24 h. DNA microarray analysis indicated that there were 25 upregulated genes and 47 downregulated genes exhibiting significantly differential expression in hippocampal neurons treated with A. bidentata extract at 1 μg/ml for 6 h when compared to those in untreated hippocampal neurons. Real-time quantitative RT-PCR and Western blot analysis demonstrated that the expression of growth-associated protein-43 in hippocampal neurons was upregulated at both mRNA and protein levels after treatment with A. bidentata extract, and the optimal dosage of the extract was also 1 μg/ml. These data confirm that A. bidentata extract could promote in vitro hippocampal neuronal growth in a dose- and time-dependent manner.     

Effects of lithium chloride on outward potassium currents in acutely isolated hippocampal CA1 pyramidal neurons

Lithium is best known for its therapeutic efficacy in the treatment of manic-depressive illness. Its clinical profile includes the antimanic and antidepressant ac- tions as well as prophylaxis of both mania and depres- sion. Despite its efficacy, the mole…     

Effect of thrombin on the apoptosis of hippocampal neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activating peptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. The numbers of apoptotic cell and apoptotic rate of hippocampal neurons treated by different concentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method and Flow Cytometry. When the concentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neurons reached peak value, were 27.3±4.0 and (29.333±4.633)%, respectively. Immunocytochemistry assay show that Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effect of thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombin induced hippocampal neuron apoptosis in a dose-dependent manner through activating protease-activated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related with the effect of high concentration thrombin induced apoptosis on hippocampal neurons. Foundation item: Supported by the Natural Science Foundation of Hainan Province (N30215) Biography: YANG Wen-qiong (1968-), female, Ph.D. candidate, research direction: cerebrovascular disease.     

Selective dendritic transport of RNA in hippocampal neurons in culture

Typical neurons of the central nervous system (CNS) elaborate tens of thousands of membrane specializations at sites of synaptic contacts on their dendrites. To construct, maintain, and modify these specializations, neurons must produce and deliver the appropriate molecular constituents to particular synaptic sites. Previous studies have revealed that polyribosomes are selectively positioned beneath postsynaptic sites, suggesting that in neurons, as in other cell types, protein synthetic machinery is located at or near the sites where particular proteins are needed. The mechanisms that deliver ribosomes and messenger RNA to their specific destinations in cells are therefore of considerable interest. Here we describe a system for RNA transport in dendrites that could provide a mechanism for the delivery of ribosomes and mRNA to synaptic sites in dendrites. Hippocampal neurons grown in culture incorporate 3H-uridine in the nucleus, then selectively transport the newly synthesized RNA into dendrites at a rate of about 0.5 mm day-1. The transport is inhibited by metabolic poisons, suggesting that it is an active, energy-dependent process. The RNA may be transported in association with the cytoskeleton.     

GABAA responses in hippocampal neurons are potentiated by glutamate

In the mammalian cortex, glutamate and gamma-aminobutyric acid (GABA) are the principal transmitters mediating excitatory and inhibitory synaptic events. Glutamate activates cation conductances that lead to membrane depolarization whereas GABA controls chloride conductances that produce hyperpolarization. Here we report that the GABAA-activated conductance in hippocampal pyramidal cells is enhanced by glutamate at concentrations below that required for its excitatory action. The GABA-potentiating effect can be induced, with comparable potency, by several glutamate analogues such as quisqualate, N-methyl-D-aspartate (NMDA), kainate and, surprisingly, by D-2-amino-5-phosphonovalerate (APV), an antagonist for NMDA receptors. Data from dose-response curves show that glutamate enhances the GABAA conductance without significantly changing GABA binding affinity. The low concentration of glutamate needed to enhance GABAA responses raises the possibility that glutamate modulates the strength of GABA-mediated transmission in the cortex.     

Glutamate activates multiple single channel conductances in hippocampal neurons

There is considerable evidence that glutamate is the principal neurotransmitter that mediates fast excitatory synaptic transmission in the vertebrate central nervous system. This single transmitter seems to activate two or three distinct types of receptors, defined by their affinities for three selective structural analogues of glutamate, NMDA (N-methyl-D-aspartate), quisqualate and kainate. All these agonists increase membrane permeability to monovalent cations, but NMDA also activates a conductance that permits significant calcium influx and is blocked in a voltage-dependent manner by extracellular magnesium. Fast synaptic excitation seems to be mediated mainly by kainate/quisqualate receptors, although NMDA receptors are sometimes activated. We have investigated the properties of these conductances using single-channel recording in primary cultures of hippocampal neurons, because the hippocampus contains all subtypes of glutamate receptors and because long-term potentiation of synaptic transmission occurs in this structure. We find that four or more distinct single-channel currents are evoked by applying glutamate to each outside-out membrane patch. These conductances vary in their ionic permeability and in the agonist most effective in causing them to open. Clear transitions between all the conductance levels are observed. Our observations are compatible with the model that all the single channel conductances activated by glutamate reflect the operation of one or two complex molecular entities.     

Potassium conductances in hippocampal neurons blocked by excitatory amino-acid transmitters

Excitatory amino acids mediate fast synaptic transmission in the central nervous system through the activation of at least three distinct ionotropic receptors: N-methyl-D-aspartate (NMDA), the alpha-amino-3-hydroxy-5-methyl-isoxasole-4-propionate (AMPA)/quisqualate (QUIS) and the kainate subtypes (for reviews, see refs 1, 2). They also activate the additional QUIS 'metabotropic' receptor (sensitive to trans-1-amino-cyclopentyl-1,3-dicarboxylate, ACPD) linked to inositol phospholipid metabolism. We have used hippocampal slice cultures to study the electrophysiological consequences of the metabotropic response. We find that activation of an ACPD-sensitive QUIS receptor produces a 'slow' excitation of CA3 pyramidal cells, resulting from depression of a Ca2(+)-dependent K+ current and a voltage-gated K+ current. Combined voltage-clamp and microfluorometric recordings show that, although these receptors can trigger an increase in intracellular Ca2+ concentration, suppression of K+ currents is independent of changes in intracellular Ca2+. These effects closely resemble those induced by activating muscarinic acetylcholine receptors in the same neurons and suggest that excitatory amino acids not only act as fast ionotropic transmitters but also as slow neuromodulatory transmitters.     

Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine

Responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) are markedly potentiated by nanomolar concentrations of glycine. This is due to the action of glycine at a novel strychnine-resistant binding site with an anatomical distribution identical to that for NMDA receptors, suggesting that the NMDA receptor channel complex contains at least two classes of amino-acid recognition site. Antagonists at the glycine-binding site associated with NMDA receptors act as potent non-competitive antagonists, but do not alter the mean open time or conductance, as estimated by fluctuation analysis. The mechanisms by which glycine acts on NMDA receptors are unknown, but single-channel recording experiments show an increase in opening frequency with no change in mean open time or conductance, suggesting that glycine could regulate transitions to states that are intermediate between binding of NMDA receptor agonists and ion-channel gating. It has been suggested that glycine acts as a co-agonist at the NMDA receptor, and that responses to NMDA cannot be obtained in the complete absence of glycine, but in these experiments the response to NMDA was measured at equilibrium, and it is unlikely that sufficient temporal resolution was achieved to detect rapid alterations in receptor gating. Using a fast perfusion system we find that glycine regulates desensitization at NMDA receptors; this has a major effect on the response to NMDA measured at equilibrium, as would occur with slower applications of agonist. Reduction of NMDA receptor desensitization by glycine provides an example of a novel mechanism for regulation of ion-channel activity.