Gas7在大鼠斜角带核发育过程中的表达

目的研究大鼠发育过程中,生长休止蛋白7(growth arrest-specific protein 7,Gas7)在斜角带核(diagonal band nucleus,DB)的表达。方法应用RT-PCR方法、Western blot方法和免疫组织化学方法观察胚胎期16.5天(E16.5)、E20.5、出生当天(P0)、生后7天(P7)、P14、P21和2月龄(成年)大鼠DB的Gas7表达及定位。结果 RT-PCR和Western blot检测显示,在E16.5时Gas7表达最弱,其后逐渐增强,至P21时达到高峰;免疫组织化学染色显示,斜角带核水平支(horizontal limb of the diagonal band,HDB)在E16.5和E20.5时出现Gas7免疫反应阳性产物,Gas7阳性神经元出现于P0,至P21时阳性神经元数量最多,染色最深;斜角带核垂直支(vertical limb ofthe diagonal band,VDB)至P14时Gas7阳性神经元最多,P21和成年均有所减少。结论 Gas7的表达在大鼠DB发育过程中具有时间和空间上的特异性,提示Gas7可能参与大鼠DB的发育过程。     

生长休止蛋白7在大鼠小脑皮质发育过程中的动态表达

目的研究生长休止蛋白7(Gas7)在大鼠小脑皮质不同发育时期的动态表达。方法采用逆转录聚合酶链反应(RT-PCR)方法检测Gas7mRNA在大鼠小脑皮质不同发育时期的表达;免疫组织化学方法观察Gas7蛋白在大鼠小脑皮质不同发育时期的表达和分布。结果 RT-PCR结果:Gas7mRNA在大鼠小脑皮质发育时期的表达呈现先增强后减弱的趋势,高峰出现在生后第21d(P21)。免疫组化实验结果:在胚胎第18.5d(E18.5)和E20.5仅Purkinje细胞层有Gas7免疫阳性产物分布;出生当天(P0)外颗粒层出现Gas7阳性神经纤维,Purkinje细胞层出现形态不规则的Gas7免疫阳性细胞;P7外颗粒层和Purkinje细胞层免疫反应增强,内颗粒层出现一些散在的Gas7强阳性细胞,胞体较小,突起清晰可见;P14小脑皮质4层均有Gas7阳性表达;P21小脑皮质3层Gas7免疫阳性反应较P14增强(P0.01);Adult(2月龄)较P21免疫反应减弱(P0.01)。结论 Gas7在大鼠小脑皮质发育过程中的动态表达呈现出时空特异性,提示Gas7基因在大鼠小脑皮质发育过程中可能起着重要的调控作用。     

小鼠海马内HDAC2的表达及其与NMDA受体、PSD-95的共定位

目的探讨组蛋白去乙酰化酶2（HDAC2）在成年C57BL/6小鼠海马内的分布及其与突触后致密区（PSD）蛋白成员的共定位,为揭示HDAC2与PSD蛋白复合物之间的内在联系及在海马相关的学习记忆过程中可能起到的调控作用提供形态学依据。方法应用免疫组化方法观察HDAC2在C57BL/6小鼠海马各区的表达分布。应用免疫荧光双标技术研究HDAC2与PSD蛋白成员N-甲基-D-天冬氨酸（NMDA）受体亚单位1（NR1）、PSD-95之间是否存在共定位。结果 HDAC2在小鼠海马CA1～CA3区锥体细胞和齿状回颗粒细胞均具有明显表达,而在各区的始层、辐射层、腔隙-分子层以及齿状回多形细胞层表达均较少。免疫荧光双标染色图片的重叠表明,HDAC2与NR1、PSD-95在小鼠海马CA1～CA3区锥体细胞层和齿状回颗粒细胞层内均可见显著共表达现象,其他区域偶见散在分布的双染神经元。结论 HDAC2在小鼠海马锥体细胞层和颗粒细胞层表达丰富,并与PSD蛋白成员间存在共定位现象。本实验结果为探讨HDAC2对谷氨酸能突触后神经元依赖的突触可塑性的调节机制提供了形态学依据。     

非基因型雌激素膜性受体GPR30在生后雌性大鼠海马内的发育学表达与亚细胞水平定位研究

为了研究非基因型雌激素膜性受体GPR30对海马的结构和功能的调节作用,应用硫酸镍铵增强显色的免疫组化技术以及酶标免疫电镜技术,观察了生后雌性大鼠海马内GPR30表达的变化及其免疫阳性产物在神经元亚细胞水平的定位情况．结果显示,GPR30免疫阳性产物主要位于海马CA区的锥体层神经元与齿状回颗粒层的神经元内,其表达水平随发育呈增加趋势．P0时在雌性大鼠海马未发现明显GPR30免疫阳性反应,P7后免疫阳性物质开始在CA2出现,P14时见于 CA1、CA2和齿状回,P30和P60主要见于CA1、CA2、CA3和齿状回．在光镜下,GPR30免疫阳性产物位于细胞核外的胞浆中,细胞核未见免疫阳性反应．在透射电镜下可见其位于神经元的胞浆内,可能主要是粗面内质网,也可见于线粒体和细胞膜．以上结果证实,GPR30是一种位于细胞核外的、非基因型作用的雌激素受体,可能参与了雌激素对海马锥体神经元突触可塑性和学习记忆等功能的调节,还可能参与了对齿状回成年神经干细胞某些活动的调节．     

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。     

甲低对新生早期大鼠海马及齿状回Goα mRNA的影响

目的：研究甲状腺功能低下（甲减）对围生早期大鼠海马及齿状回Goα mRNA表达的影响。方法：采用地高辛标记寡核苷酸探针原位杂交技术,观察7d龄围生期甲减及正常Wistar大鼠海马CA1－4区及齿状回Goα mRNA的表达状况。结果：7d龄甲状腺激素对围生早期海巴及齿状回Goα 基因的表达具有负调节作用。     

微管相关蛋白tau在不同年龄大鼠海马内的定位分布特点

目的 检测微管相关蛋白tau和Ser396/404位点磷酸化tau在胚胎期大鼠和出生后直至成熟期大鼠海马内的表达及变化规律,浅析与神经细胞分裂和分化的关系.方法 用免疫组织化学SABC法显示孕18d、出生后1d、1w、2w、2m的大鼠脑冠状切面总tau（R134d）、Ser396/404位点磷酸化tau（PHF-1）的表达.结果 ①胚胎期大鼠海马CA区的锥体细胞数量明显多于出生后,随着脑的发育,锥体细胞层的神经细胞数量逐渐下降;②孕18d大鼠海马内有丰富的总tau和PHF-1 tau的表达,并且海马内各区的阳性物质表达均匀,生后1d和1w两种物质表达仍然很强,阳性产物主要分布在海马CA1和CA2区以锥体细胞轴突为主要成分的始层,在锥体细胞树突集中的分子层和胞核内也有少量分布,而在生后2w和2m大鼠海马内各区均未见密集的阳性产物表达.结论 不同年龄大鼠海马内总tau和PHF-1 tau的表达和分布变化可能与神经系统发育过程中细胞的分裂和分化有关.     

AMPA受体和相关蛋白在束缚应激大鼠相关脑区的表达变化及逍遥散对其影响

目的：观察海马及杏仁核α-氨基羟甲基恶唑丙酸（AMPA）受体亚基和相关调节蛋白在束缚应激状态下蛋白表达变化及逍遥散的调节作用。方法：使用每天捆绑3 h的方法制作慢性束缚应激动物模型,并用逍遥散进行干预,分别于7 d后和21 d后用Western blot方法检测各组大鼠海马CA1区、CA3区、齿状回（DG）和杏仁核的AMPA受体亚基GluR2/3及N-乙基顺丁烯二酰亚胺敏感性的融合蛋白（NSF）、PKC作用蛋白1（PICK1）蛋白表达的情况。结果：7 d应激可使DG和杏仁核的GluR2/3、NSF表达显著降低（P均〈0.05）,使PICK1在CA1区的表达量显著增多（P〈0.05）,逍遥散对PICK1变化显示出一定调节作用。21 d应激可使CA1区的GluR2/3、NSF表达升高,其中GluR2/3有显著性差异（P〈0.01）,而在杏仁核表达有降低趋势,逍遥散对其均有显著调节作用（均为P〈0.05）,21 d应激使杏仁核PICK1表达量出现升高趋势,逍遥散可显著降低其表达（P〈0.05）。结论：AMPA受体在短期重复应激和慢性应激状态下反应不同,海马和杏仁核反应相反,逍遥散对慢性应激状态下AMPA受体表达的调节作用较短期重复应激强。     

Gas7在成年大鼠脊髓和脊神经节的表达

目的 研究生长休止蛋白7（Gas7）在成年大鼠脊髓和脊神经节的表达.方法 成年SD大鼠12只,采用逆转录聚合酶链反应（RT-PCR）方法、焦油紫染色以及免疫组织化学方法来观察Gas7基因核酸和蛋白在成年SD大鼠脊髓和脊神经节的表达.结果 RT-PCR结果显示,脊髓和脊神经节有较丰富的Gas7 mRNA表达.免疫组化结果显示：与焦油紫染色相对照,脊髓灰质各板层神经元均表达Gas7蛋白,与其它版层相比较,后角Ⅱ版层胶状质的小细胞和前角Ⅸ版层的运动神经元显色较深且数量较多.脊髓白质Gas7免疫阳性反应较弱且分布均匀.脊神经节内大型感觉神经元呈Gas7免疫强阳性反应,中、小型感觉神经元为弱阳性反应.结论 本文首次描述了Gas7在成年大鼠脊髓和脊神经节的表达,为进一步研究Gas7在成年神经系统再生和修复过程中的功能提供形态学基础.     

AVP_（4－8）结合点在大鼠海马内的分布和AVP_（4－8）对其受体发育的影响：放射自显影研究

本文利用放射自显影方法结合神经毒对海马神经元的选择性损毁观察ＡＶＰ（４－８）结合点在大鼠海马内的分布和定位;利用外源性ＡＶＰ（４－８）对新生大鼠的处理,观察海马ＡＶＰ（４－８）结合点的发育调节。在成年大鼠海马内,ＡＶＰ（４－８）结合点集中分布在整个海马的锥体细胞层和齿回的颗粒细胞层。秋水仙碱处理后,齿回颗粒细胞层消失,齿回区的ＡＶＰ（４－８）结合点也消失。红藻氨酸（Ｋａｉｎｉｃａｃｉｄ）处理后海马ＣＡ３－ＣＡ４的锥体细胞层消失,该区的ＡＶＰ（４－８）结合点也消失。新生大鼠海马锥体细胞层的ＡＶＰ（４－８）结合点在出生后第６天开始出现,齿回颗粒细胞层的ＡＶＰ（４－８）结合点在出生后第７天开始出现。然而,新生大鼠每天经外源性ＡＶＰ（４－８）处理,海马锥体细胞层和齿回颗粒细胞层的结合点均在出生后第５天已变得十分稠密。本文就大鼠海马ＡＶＰ（４－８）结合点的特异性分布和ＡＶＰ（４－８）处理促进海马ＡＶＰ（４－８）结合点的发育与成年后大鼠学习能力的提高的相互关系作了讨论。     

Cre-mediated cerebellum- and hippocampus-restricted gene mutation in mouse brain

Using the phage P1-derived Cre/loxP recombination system, we have created a line of cre-transgenic mice in which the Cre-mediated gene deletion is restricted to granule cells of cerebellum and dentate gyrus of hippocampus. Low levels of deletion were also present in pyramidal cells of hippocampal CA1 and CA3 fields. The Cre/loxP recombination occurred prenatally. The recombination efficiencies in the granular layer of the cerebellum, the granular layer of the dentate gyrus, and the CA1 and CA3 pyramidal cells of the hippocampus were 34.0%, 23.1%, 3.0%, and 9.8%, respectively. This line of cre-transgenic mice should be conducive to studies of the effect of a gene mutation upon brain development and plasticity.     

Gene expression in developing rat hippocampal pyramidal neurons appears independent of mossy fiber innervation.

In the rat, neonatal gamma-irradiation of the hippocampus induces a selective destruction of dentate granule cells and prevents the development of the mossy fiber-CA3 pyramidal cell connection. In the absence of mossy fiber input, the CA3 pyramidal neurons exhibit morphological alterations and rats deprived of dentate granule cells fail to develop kainate-induced epileptic activity in the CA3 pyramidal neurons. Neonatal elimination of the granule cells also impairs learning and memory tasks in adult rats. In the present work, we assessed by in situ hybridization and semi-quantitative RT-PCR, whether in the pyramidal layers, the absence of mossy fiber input alters the expression of a number of genes involved in activity-dependent signal transduction, in GABAergic neurotransmitter signaling and in neurite development via microtubule organization. Surprisingly, we show that the expression and the developmentally regulated alternative splicing of the genes we examined in the developing hippocampus are not altered in the pyramidal neurons, whether the dentate granule afferents are present or absent. Our results suggest that in the CA3 pyramidal layer, the developmental expression patterns of the mRNAs we studied are independent of extrinsic cues provided by mossy fiber input.     

Decreased Insulin-Like Growth Factor-I and Its Receptor Expression in the Hippocampus and Somatosensory Cortex of the Aged Mouse

Insulin-like growth factor-I (IGF-I) is a multifunctional polypeptide and has diverse effects on brain functions. In the present study, we compared IGF-I and IGF-I receptor (IGF-IR) immunoreactivity and their protein levels between the adult (postnatal month 6) and aged (postnatal month 24) mouse hippocampus and somatosensory cortex. In the adult hippocampus, IGF-I immunoreactivity was easily observed in the pyramidal cells of the stratum pyramidale in the hippocampus proper and in the granule cells of the granule cell layer of the dentate gyrus. In the adult somatosensory cortex, IGF-I immunoreactivity was easily found in the pyramidal cells of layer V. In the aged groups, IGF-I expression was dramatically decreased in the cells. Like the change of IGF-I immunoreactivity, IGF-IR immunoreactivity in the pyramidal and granule cells of the hippocampus and in the pyramidal cells of the somatosensory cortex was also markedly decreased in the aged group. In addition, both IGF-I and IGF-IR protein levels were significantly decreased in the aged hippocampus and somatosensory cortex. These results indicate that the apparent decrease of IGF-I and IGF-IR expression in the aged mouse hippocampus and somatosensory cortex may be related to age-related changes in the aged brain.     

Changes of hippocampal neurons after perinatal exposure to ethanol

The effect of ethanol on the structural development of the central nervous system was studied in offspring of Wistar rats, drinking 20 % ethanol during pregnancy and till the 28th day of their postnatal life. The structural changes in the hippocampus and dentate gyrus were analyzed at the age of 18, 35 and 90 days. A lower width of pyramidal and granular cell layers, cell extinction and fragmentation of numerous nuclei were found in all experimental animals compared to control animals. The extent of neural cell loss was similar in all monitored areas and in all age groups. At the age of 18 and 35 days, the degenerating cells were observed in the CA1 and CA3 area of the hippocampus and in the ventral and dorsal blade of the dentate gyrus. Numerous glial cells replaced the neuronal population of this region. Some degenerating cells with fragmented nuclei were observed at the age of 90 days. Our experiments confirmed the vulnerability of the developing central nervous system by ethanol intake during the perinatal period and revealed a long-lasting degeneration process in the hippocampus and dentate gyrus.     

Death Receptor 5 and Neuroproliferation

Tumor necrosis factor-related apoptosis-inducing ligand or Apo2 ligand is a member of the tumor necrosis factor superfamily of cytokines that induces apoptosis upon binding to its death domain-containing transmembrane receptors, death receptors 4 and 5 (DR4, DR5). However, DR5 is also expressed in the developing CNS where it appears to play a role unrelated to apoptosis, and instead may be involved in the regulation of neurogenesis. We report on the distribution of DR5 expression in mouse hippocampus, cerebellum, and rostral migratory stream (RMS) of olfactory bulb from embryonic (E) day 16 (E16) to postnatal (P) day (P180). At E16, DR5-positive cells were distributed widely in embryonic hippocampus with strong immunostaining in the developing dentate gyrus. In newborn hippocampus, DR5-positive cells were predominantly located in proliferative zones, such as dentate gyrus, subventricular zone, and RMS. After postnatal day 7 (P7), the number of DR5-positive cells decreased, and cells with intense fluorescence were primarily restricted to the subgranular layer (SGL), although the granular cell layer showed weak fluorescence. After P30, only few DR5-positive cells were found in SGL, and mature granule cells were negative for DR5 expression. To address whether DR5 expression is a restricted to progenitor cells and newborn neurons, we performed 5-bromo-deoxyuridine labeling. We report that proliferative cells in the SGL selectively express DR5, with lower levels of expression in cells positive for doublecortin, a marker of newborn neurons. In addition, the stem cells in intestine, cerebellum, and RMS were also demonstrated to be DR5-positive. In the meantime, in cerebellum, DR5-positive cells were also positive for glial fibrillary acidic protein, a marker of proliferative Bergmann cells. We conclude that DR5 is selectively expressed by neuroprogenitor cells and newborn neurons, suggesting that the DR5 death receptor is likely to play a key role in neuroproliferation and differentiation.