一种获取精确胚胎天数鼠脑皮质神经细胞原代培养方法

目的:探讨获取精确胚胎天数鼠脑皮质神经细胞原代培养方法.方法:首先将性成熟雌雄SD大鼠1∶1合笼于代谢饲养盒内,次日观察有无阴栓(同时做阴道涂片),若有记为E0.取E17孕鼠断颈处死,无菌状态下取出子宫,剥离羊膜与胚外膜,取得胚胎.胚胎断头,在解剖显微镜下用尖镊沿双眼、小脑和脑干腹侧切取大脑皮质,用0.125％胰酶进行温和消化,采用MEM加10％胎牛血清、10％马血清和1％ L-Glutamine混合培养基,用0.73 MPoly-L-Omithine Hydrobromide处理的培养板入37℃,10％CO2孵箱内培养.结果:雌鼠一次可排出阴栓3-5个不等,阴栓为奶黄色、锥体形、质韧.同时做阴道涂片可见活动精子;获取得胚胎脑皮质细胞呈贴壁生长,其胞体饱满,折光性好,突起明显,与周围细胞形成网络连接.结论:用阴栓并精子涂片判断胚胎胎龄简便而准确;E17胚胎适宜体外培养获得脑皮质细胞;组合培养基加处理过的培养板和10％ CO2孵箱浓度的新方法获得了状态良好的脑皮质神经细胞.     

经颅磁刺激在大脑皮质研究中的应用和进展

经颅磁刺激（TMS）是一种能够在脑中感应聚焦电流,瞬间调制大脑皮质的无创方法,在临床研究、基础神经学和诊治疾病等方面有许多应用。通过记录运动皮质诱发电位（MEPs),TMS已经或将成为探测脑下运动路径传导、评价皮质兴奋性、皮质映射和研究皮质塑性的常规工具。TMS能够主动干预脑功能,这种特性使它成为研究正常人脑－行为关系的独特技术,可以建立脑活动与任务完成之间的因果关系,探索脑功能连接。近年来的许多实验又表明,TMS在运动紊乱和精神疾病方面有潜在的治疗作用,但达到临床应用还有一定距离。     

Fos蛋白在脑挫伤皮质和海马中表达的研究

目的探讨脑挫裂伤后Fos因蛋白在皮质和海马中表达的变化.方法取成年大鼠45只分为正常对照组、实验对照组和模型组.用Feemey's自由落体法将其中32只复制脑挫伤动物模型;脑挫伤后30min、1h、2h、4h、8h、12h、24h和48h,分别取脑;石蜡切片,免疫组织化学染色.结果正常对照组脑内无Fos阳性细胞;实验对照组局部皮质有Fos微弱表达;模型组脑挫伤侧皮质和海马有Fos阳性表达.30min后已有阳性细胞;2～4h达高峰,阳性细胞多,而且着色较深,8～24h逐渐减弱,48h则不见阳性细胞.结论脑创伤可以导致伤侧皮质和海马c-fos基因蛋白过表达,而且各时间段表达强度不同,观察c-fos基因表达强度,可以作为临床提示脑部受伤时间的参考指标.     

ERK1/2和p-ERK1/2在肺癌组织中的表达及意义

目的研究细胞外信号调节激酶1/2（extracellular regulated kinase 1/2,ERK1/2）及其磷酸化状态（p-ERK1/2）在不同分化程度肺癌中的表达情况,探讨二者与肺癌侵袭、转移的关系。方法采用免疫组化（Envision）法,检测79例肺癌组织及l2例癌旁正常肺组织中ERK1/2和p-ERK1/2的表达。结果ERK1/2在高、中、低分化组表达率分别为13.6%,39.4%,66.7%,p-ERK1/2在高、中、低分化组表达率分别14.3%,27.3%,79.2%（P〈0.05）;无淋巴结转移者阳性率为20%,有淋巴结转移者阳性率为50.1%（P〈0.05）。ERK1/2和p-ERK1/2的表达在不同年龄、性别、肿瘤大小、肿瘤病理类型无显著性差异,而与分化程度有关,其中p-ERK1/2的表达还与有无淋巴结转移有关。结论ERK1/2和p-ERK1/2在肺癌组织中高表达且与分化程度有关。     

ERK1/2在三倍体湘云鲫组织及胚胎中的表达分析

在细胞信号网络中丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号传递途径起着极为重要的作用,控制着细胞多种生理过程,如细胞生长、发育、分裂、死亡等.ERK是MAPK家族重要的成员,通过检测三倍体湘云鲫胚胎和成体组织中ERK1/2的表达情况,初步研究ERK在鱼类发育中的作用.蛋白免疫杂交结果显示ERK1/2在三倍体湘云鲫的胚胎发育各个时期和组织中均有较高的表达,该结果表明ERK在鱼类发育的过程中发挥重要的作用.     

非侵入性脑刺激技术在工作记忆研究中的应用

工作记忆是人类认知功能的核心组成部分,负责信息的短暂存储与加工,并在日常任务执行中发挥重要作用。工作记忆的缺陷会导致高级认知过程和发展障碍,并且随着个体年龄的增长,工作记忆能力往往会衰退。鉴于大脑具有可塑性的特点,非侵入性脑刺激（noninvasive brain stimulation,NIBS）已经被用于激活特定大脑区域,以改善工作记忆功能。目前的证据表明,NIBS有潜力成为改善工作记忆的有效工具,主要包括经颅磁刺激（transcranial magnetic stimulation,TMS）、经颅电刺激（transcranial electrical stimulation,tES）等。本文首先综述了工作记忆的神经生理基础,然后回顾了近年来 NIBS 在健康成人工作记忆干预中的应用,讨论了其在提升工作记忆方面的潜在效益和局限,为工作记忆的深入研究和临床转化提供一定参考。     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.     

大鼠内侧前额叶皮质内ERK1/2 活化参与脊髓损伤后抑郁情绪

目的:观察细胞外信号调节激酶1/2(ERK1/2)的活化在脊髓损伤引起抑郁中的作用。方法:应用Western blot和行为药理学方法,观察脊髓损伤后(SCI)大鼠内侧前额叶皮质内(mPFC)ERK1/2及磷酸化-ERK1/2(p-ERK1/2)的表达情况及ERK1/2磷酸化抑制剂U0126对抑郁样行为的影响。结果:脊髓损伤后的第2天到第8周,SCI模型大鼠的BBB评分均显著低于假手术组,差异具有统计学意义(p0.05)。脊髓损伤后8周-12周,SCI模型大鼠强迫游泳不动时间与假手术组相比明显缩短,mPFC内pERK1/2蛋白表达水平明显升高,总ERK 1/2的蛋白水平则未见组间差异,而给予U0126的大鼠的不动时间与给药之前相比明显延长增加,mPFC内pERK1/2蛋白表达水平较SCI模型大鼠明显降低,差异均具有统计学意义(P0.05)。结论:内侧前额叶皮质内ERK1/2的激活参与了脊髓损伤后引起的突触可塑性,在相关的抑郁样行为的产生中发挥了重要的作用。     

Gas7在大鼠梨状皮质发育过程中的表达

目的研究生长休止蛋白(Growth arrest-specific protein 7,Gas7)在大鼠梨状皮质发育过程中的表达。方法采用逆转录聚合酶链反应(RT-PCR)方法和免疫组织化学方法检测Gas7核酸与蛋白在SD大鼠胚胎第18.5天(E18.5)、E20.5、出生当天(P0)、生后第7天(P7)、P14、P21和成年(Adult)各时期梨状皮质中的表达。结果 RT-PCR结果显示Gas7核酸在大鼠梨状皮质各发育时期均有表达,在P14时表达最强;免疫组织化学方法显示梨状皮质在E18.5时即出现Gas7免疫阳性产物,至P7时出现清晰的Gas7免疫阳性细胞,至P14时细胞数达到峰值,免疫阳性反应最强,P21细胞数少于P14(P0.05),Adult细胞数少于P21(P0.05)。结论 Gas7在梨状皮质的表达具有时间上的差异性,提示Gas7可能在梨状皮质结构形成和功能成熟方面起着重要的调控作用。     

大鼠海马神经细胞VDAC1基因shRNA慢病毒表达载体的构建及干扰效果评价

目的:构建线粒体电压依赖性阴离子通道(VDAC1)短发卡样RNA重组慢病毒(LV)载体,沉默海马神经细胞VDAC1基因表达,观察所构建的慢病毒载体对海马神经细胞的干扰效果。方法:根据基因库提供的大鼠VDAC1基因的核苷酸序列(序列号为：AB039662.1),设计合成3条LV3-VDAC1-shRNA及1条无义的阴性表达载体;将构建好的慢病毒载体转染到海马神经细胞,按照不同的转染条件对细胞分为空载组、对照(NC)组、病毒组,分别在病毒感染复数(MOI)值在100和10的条件下进行转染;采用荧光定量PCR和Western blot的方法分别检测VDAC1基因在mRNA水平和蛋白水平的表达。结果:与空载组相比,慢病毒感染组的扩增倍数均大于1,VDAC1基因在mRNA水平与蛋白水平表达均明显升高(P<0.05);与对照组相比,病毒感染复数(MOI)=10与MOI=100之间没有显著差异(P>0.05);PCR结果与Western blot结果综合来看,VDAC1转录水平表达越高,蛋白水平表达越低,呈现反向关联的状态。结论:三种慢病毒对海马神经细胞都有干扰效果,VDAC1基因的mR...     

新生BALB/c小鼠大脑皮质神经元细胞培养方法的建立

目的:经改良和优化,建立高纯度BALB/c小鼠大脑皮质神经元培养的方法.方法:采用L-多聚赖氨酸包被细胞培养板,取新生BALB/c小鼠(出生24 h内)大脑皮质组织,经0.25%胰酶消化后吹打成单个细胞,按1×106/孔接种于35 mm的六孔板中,用神经元细胞培养种植液培养6 h后换神经元细胞培养饲养液,培养40 h时...     

Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca²⁺ concentration. Involvement of the Ca_v2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Ca_v2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling cascade. This finding may provide further understanding of the mode of berberine action in the brain and highlights the therapeutic potential of this compound in the treatment of a wide range of neurological disorders.     

Glutamate-Induced Increase in Intracellular Ca2+ in Cerebral Cortex Neurons Is Transient in Immature Cells but Permanent in Mature Cells

The free cytosolic Ca2+ concentration ([Ca2+]i) of cultured cerebral cortex neurons was determined using a fluorescent Ca2+ chelator (Fluo-3) after exposure of the neurons to glutamate. Mature neurons (8 days in culture) responded within 45 s to 100 microM glutamate by an increase in [Ca2+]i from 75 to 340 nM, an increase that during the following 6 min of exposure reached 400 nM. This increase in [Ca2+]i could not be reversed by removal of glutamate. In the absence of extracellular CaCl2, only part of the initial, rapid, glutamate-induced increase in [Ca2+]i was observed in these neurons. In contrast to these findings, neurons cultured for only 2 days (immature neurons) exhibited only a small (from 75 to 173 nM) increase in [Ca2+]i after exposure to 100 microM glutamate, and this rapid increase in [Ca2+]i tended to decline on prolonged exposure to glutamate. Moreover, after removal of glutamate, the increase in [Ca2+]i was fully reversible. Pharmacological characterization of the response to glutamate in mature neurons showed that the N-methyl-D-aspartate (NMDA) receptor antagonists phencyclidine and D-2-amino-5-phosphonovalerate phosphonovalerate blocked 75 and 90%, respectively, of the response, whereas the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione had little effect.     

Differential Regulation of GABAA Receptor Gene Expression by Ethanol in the Rat Hippocampus Versus Cerebral Cortex

Abstract: Previous research has shown that chronic ethanol consumption dramatically alters GABA_A receptor α₁ and α₄ subunit gene expression in the cerebral cortex and GABA_A receptor α₁ and α₆ subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABA_A receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABA_A receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABA_A receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABA_A receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABA_A receptor α₄ subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABA_A receptor α₁, α₂, α₃, α_2/3, or γ₂ was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABA_A receptor α₄ subunits and significantly decreased the relative expression of cerebral cortical GABA_A receptor α₁ subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABA_A receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.     

新生SD大鼠皮质神经元的体外培养和鉴定

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24h内的新生SD大鼠皮质,用木瓜酶和DNaseⅠ共同消化,5％胎牛血清终止消化,吹打分离组织获得单细胞悬液,进行细胞计数,用无血清DMEM／F12种植培养,4h后换成用无血清Neurobasal配制的维持培养液继续培养,尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10d,神经元胞体饱满,结构清晰完整,光晕明显,折光性强,可见粗长的树突和轴突,相邻细胞形成紧密网状联系,神经元纯度达到96％以上。结论：经改良和优化,无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。     

The Extrusion of Sodium Ions from Presynaptic Nerve Endings of Rat Cerebral Cortex

(Na⁺-K⁺) ATPase is present in synaptosomal preparations and it is assumed to represent the sodium-potassium pump. 10 μm -noradrenaline activates (Na⁺-K⁺) ATPase approximately 100%, but 50 μm -noradrenaline does not stimulate the rate of ²²Na extrusion from synaptosomes. The results suggest that it is unlikely that the noradrenaline stimulation of (Na⁺-K⁺) ATPase is part of a feedback mechanism whereby released noradrenaline can influence the activity of the presynaptic sodium pump.     

Modulation of Antioxidant Enzyme Expression by PTU-Induced Hypothyroidism in Cerebral Cortex of Postnatal Rat Brain

This study aimed to elucidate the effect of 6-n-propylthiouracil (PTU)-induced hypothyroidism on oxidative stress parameters and expression of antioxidant enzymes in cerebral cortex of rat brain during postnatal development. A significant decrease in levels of lipid peroxidation and H₂O₂ were seen in 7 and 30 days old PTU-treated rats with respect to their controls. Significantly decreased activities of superoxide dismutase (SOD) and catalase (CAT) along with the translated products of SOD1 and SOD2 were observed in 7, 15 and 30 days old PTU-treated rats as compared to their respective controls. However, increase in translated product of CAT was seen in all age groups of PTU-treated rats. Glutathione peroxidase activity was decreased in 7 days and increased in 15 days old PTU-treated rats with respect to their control groups. Histological sections clearly show a decline in neuronal migration with neurons packed together in the hypothyroid group as compared to the control.     

成年哺乳动物皮质区神经发生的研究进展

神经发生是神经干细胞在适当的条件下分化成功能性整合神经元的过程,主要包括细胞的增殖、迁移、分化和存活。成年神经发生区以前脑室管膜下区(Subventricular zones,SVZ)和海马齿状回颗粒层下区(Subgranular zones,SGZ)为主,但皮质作为神经元和神经胶质细胞数量最多、分布最广,同时也是哺乳动物高度发展的脑区,是否有成年神经元新生,已成为近年来神经科学领域的研究热点[1,2]。现本文就未成熟神经元在皮质区的研究方法、分布、来源与转归、病理生理功能影响等方面探讨成年哺乳动物皮质神经发生现象。