Expression of calcium/calmodulin-dependent protein kinase Ⅱ in the hippocampal subregions of the rat during postnatal development

目的 探讨Wistar大鼠生后海马发育过程中钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达.方法 应用免疫荧光方法检测CaMKⅡ在生后不同时期大鼠海马CA1、CA3区和齿状回(DG)中的表达情况(n=48). 结果 CaMKⅡ于生后各期海马CA1区和DG的表达逐渐增强,生后第10天(P10)达高峰期,此后逐渐减弱;于CA3区的表达在P4和P10时均较高.其中,CaMKⅡ在CA3区的表达高于在CA1区和DG的表达,在多形层和分子层的表达高于在锥体细胞层或颗粒细胞层的表达. 结论 CaMKⅡ在CA1、CA3区和DG中的表达具有特异性的时空分布模式,这可能与其在生后发育过程中的突触发生,树突、轴突形成,海马的成熟以及学习记忆功能相关.     

钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠生后海马发育中的表达

目的 探讨Wistar大鼠生后海马发育过程中钙/钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）的表达。 方法 应用免疫荧光方法检测CaMKⅡ在生后不同时期大鼠海马CA1、CA3区和齿状回（DG）中的表达情况（n =48）。结果 CaMKⅡ于生后各期海马CA1区和DG的表达逐渐增强，生后第10天（P10）达高峰期，此后逐渐减弱；于CA3区的表达在P4和P10时均较高。其中，CaMKⅡ在CA3区的表达高于在CA1区和DG的表达，在多形层和分子层的表达高于在锥体细胞层或颗粒细胞层的表达。结论 CaMKⅡ在CA1、CA3区和DG中的表达具有特异性的时空分布模式，这可能与其在生后发育过程中的突触发生，树突、轴突形成，海马的成熟以及学习记忆功能相关。     

NR1、NR2A与PSD-95在生后大鼠海马发育中的表达

目的:探讨N-甲基-D-天冬氨酸受体亚单位1(N-methy1-D-aspartate receptor subunit1,NR1)、N-甲基-D-天冬氨酸受体亚单位2A(N-methy1-D-aspartate receptor subunit2A,NR2A)与突触后密度蛋白-95(Postsynapticdensity protein 95,PSD-95)在Wistar大鼠海马生后发育过程中的表达。方法:应用免疫荧光染色方法检测NR1、NR2A与PSD-95在生后不同时期大鼠海马CA1、CA3区和齿状回(DG)中的表达情况。结果:NR1于生后各期海马CA1、CA3区和DG的表达均增强,P14~P21达高峰期后减弱。NR2A于生后在海马CA1和CA3区的表达略有减弱,P4后增强至高峰期P14,然后轻微减弱,在DG中NR2A的表达生后略有减弱,P4后在增强的趋势中于P7、P14和P28后均有不同程度的减弱,高峰期在P28。PSD-95于生后各期海马CA1、CA3区和DG的表达均增强,P21~P28达高峰期后轻微减弱。结论:NR1、NR2A和PSD-95在CA1、CA3区和DG中的表达具有特异的时空分布模式,此模式可能与其在生后发育中发挥的不同生理功能相关。     

APPSWE转基因小鼠发育过程中海马CA1区神经细胞凋亡研究

目的 探讨APPSWE转基因小鼠发育过程中海马CA1区神经细胞凋亡规律.方法 取不同发育时间(P0、P7、P14、P30、P90、P180)APPSWE转基因模型鼠与同时间点对照鼠,Nissl染色观察海马结构和锥体细胞形态,免疫组织化学方法观察海马细胞内Caspase-3表达变化,RT-PCR检测Caspase-3 mRNA表达变化.结果 随着小鼠的生长发育,P14时间点以后,模型组CA1区神经元Caspase-3阳性细胞密度比对照组高,RT-PCR检测结果与Caspase-3免疫组织化学结果基本一致.结论 APPSWE转基因小鼠发育中的海马神经细胞过度凋亡可能与阿茨海默病的发生、发展具有联系.     

生后大鼠海马CA区CXCR4蛋白的表达变化

目的:研究CXCR4蛋白在生后大鼠海马CA区的表达变化.方法:用免疫印迹法和免疫组织化学法定量、定位检测CXCR4在海马CA区的表达变化.结果:从生后4d到成年大鼠海马CA区均可见CXCR4蛋白表达阳性的细胞,其胞质呈棕黄色.生后早期,可见CA区锥体细胞伸出的轴丘上有棕黄色显色;后期其长轴突及短树突上也有不同程度CXCR4蛋白表达.免疫印迹检测显示,生后各时间点在分子量为43kD处均检测到CXCR4阳性条带.CXCR4在CA区的相对表达量于生后1周达高峰,随年龄增长逐渐下降,至生后3周CXCR4蛋白表达降至成年水平.结论:CXCR4蛋白在大鼠海马CA区的表达具有时空性差异,提示CXCR4参与了大鼠海马生后发育过程,并可能与不同时期海马结构和功能的发育有关.     

线粒体凋亡路径参与创伤后应激障碍大鼠海马神经元凋亡的调控

目的观察Caspase-9、Caspase-3和细胞色素C(CytC)在创伤后应激障碍(PTSD)大鼠海马神经元中的表达及相互关系,探讨PTSD大鼠海马神经元凋亡的信号转导机制。方法成年雄性Wistar大鼠60只,采用国际认定的无连续单一应激(SPS)方法刺激大鼠建立PTSD大鼠模型,取SPS刺激后1d、4d、7d、14d、28d组和正常对照组。应用免疫组织化学、免疫荧光和免疫印迹法检测Caspase-9、Caspase-3和CytC蛋白的表达。结果免疫组织化学和免疫荧光结果显示,CytC蛋白于SPS刺激后4d在海马神经元胞质内的表达达到高峰,并维持较高水平,SPS刺激后7d逐渐下降。SPS刺激后,Caspase-9和Caspase-3阳性表达增强,均于SPS刺激后7d达到高峰。免疫印迹结果显示,与正常对照组相比,模型组海马胞质内CytC蛋白水平明显上调,于SPS刺激后4d达到较高水平。而SPS刺激后线粒体中CytC蛋白水平则显著下降。在正常对照组,无Caspase-9和Caspase-3活性片段;在模型组,出现Caspase-9和Caspase-3活性片段,两者均于SPS刺激后7d达到高峰,14d开始下调。结论神经元凋亡可能是导致PTSD大鼠海马萎缩的重要机制之一;线粒体通路的激活参与了PTSD大鼠海马神经元凋亡的调控。     

小鼠海马发育中Bcl-2和Bax表达的体视学观察

目的探讨B淋巴细胞瘤-2基因(Bcl-2)和促凋亡基因Bax(Bax)在C57/BL6小鼠海马发育过程中的表达变化。方法取胚龄(E)18、20 d和生后(P)1、3、7、14、21、28 d以及2、3、6、15、18个月的C57/BL6小鼠海马,每组8只,应用免疫组织化学技术及体视学方法检测Bcl-2和Bax蛋白的表达。结果 E18 d~P21 d,在齿状回(DG)的颗粒层、海马阿蒙角(CA)以及CA各区(CA1~CA4)的锥体层,Bcl-2和Bax阳性细胞及其体密度均呈现出先逐渐增加再逐渐降低的趋势,除DG区Bax阳性细胞及其体密度在P14 d达到最高外,其他均在P7 d达到最高(P0.01)。P28 d后均趋于稳定(P0.05)。CA锥体层及DG颗粒层Bcl-2/Bax的体密度比值在P1d显著降低(P0.01),之后趋于稳定(P0.05)。结论小鼠海马Bcl-2和Bax的表达在胚胎发育晚期和生后发育早期较多,而在成年及老年期的表达稳定在较低水平,它们可能参与了海马的塑形过程。     

β-淀粉样前体蛋白转基因小鼠发育过程中海马CA1区神经细胞凋亡变化

目的：探讨瑞典家系β-淀粉样前体蛋白（APPSWE）转基因小鼠发育过程中海马CA1区神经细胞凋亡规律。方法：取不同发育时间（P0、P7、P14、P30、P90、P180）APPSWE转基因模型鼠与同时间点对照鼠，Nissl染色观察海马结构和锥体细胞形态，免疫组织化学方法观察海马细胞内Caspase-3表达变化，RT-PCR检测Caspase-3mRNA表达变化。结果：随着小鼠的生长发育，P14时间点以后，模型组CA1区神经元Caspase-3阳性细胞密度比对照组高；RT-PCR检测结果与Caspase-3免疫组织化学结果基本一致。结论：APPSWE转基因小鼠发育中的海马神经细胞过度凋亡可能与阿尔茨海默病的发生、发展具有联系。     

Caspase-9在生后小鼠海马发育中的表达

目的观察小鼠海马发育过程中凋亡相关基因caspase-9的表达变化。方法取出生后1d、3d、7d、14d、21d和28d的小鼠海马,应用免疫组织化学技术、免疫印迹技术及图像分析技术检测caspase-9的表达变化。结果 P1 d海马CA区锥体细胞层和齿状回颗粒细胞层caspase-9阳性表达产物平均光密度最高,P3 d~P21 d caspase-9阳性表达产物平均光密度逐渐降低,P28表达水平最低。结论凋亡相关基因caspase-9在生后小鼠海马发育过程表达水平呈逐渐降低的趋势。     

大鼠创伤性脑损伤后细胞凋亡及NOS阳性细胞的变化

目的：探讨大鼠创伤性脑损伤后不同时相皮质、海马、隔区细胞凋亡及NOS、ChAT阳性细胞的变化。方法：采用大鼠自由落体脑损伤模型，伤后1、2、3、4、5、7、10d取脑切片，经Nissl染色，用TUNEL法检测细胞凋亡，NADPH—d组化染色观察NOS阳性细胞，ChAT免疫组化染色观察隔区ChAT阳性细胞。结果：Nissl染色可见损伤侧海马CA2、CA3区锥体细胞层细胞消失或紊乱。损伤区周围皮质凋亡细胞伤后3d达到高峰；损伤侧海马凋亡细胞伤后5d达到高峰；损伤侧隔区凋亡细胞7d达到高峰。正常侧上述脑区各时相点均未见到凋亡细胞。损伤区周围皮质、损伤侧海马和隔区iNOS阳性细胞数量明显增加。损伤侧隔区ChAT阳性神经元也明显减少。结论：大鼠创伤性脑损伤后损伤区周围皮质和损伤侧海马、隔区细胞凋亡数量的变化与伤后时程有关。伤后细胞iNOS表达增加是导致细胞凋亡的因素。     

Spatiotemporal Expression of HDAC2 during the Postnatal Development of the Rat Hippocampus

Background: Histone acetylation, which is a chromatin modification of histone tails, can dynamically regulate the expression of various genes in normal development. HDAC2 is a negative regulatory factor of acetylation and closely related to learning and memory. NSE is a nerve marker and vital for maintaining physiological functions in nervous system. Currently, few studies associated with the expression pattern of HDAC2 in postnatal rat hippocampus have been reported. This study aimed to explore the temporal and spatial expression pattern of HDAC2, helping to reveal the expression characteristics of HDAC2 during postnatal neuronal maturation. Materials and Methods: With NSE as a biomarker of neuronal maturation at postnatal days 1, 3, 7 and weeks 2, 4, and 8 (P1D, P3D, P7D, P2W, P4W, P8W), the expression patterns of HDAC2 in rat hippocampus were examined using real-time PCR and western blotting. Additionally, the subcellular distribution of HDAC2 was analysed by immunofluorescence. Results: We found that HDAC2 was highly expressed in the neonatal period and decreased gradually. HDAC2 expression was widely distributed in neurons of hippocampal CA1, CA3 and DG regions and gradually shifted from the nucleus to the cytoplasm during postnatal development. Altogether, the expression of HDAC2 decreased gradually with different subcellular localizations throughout development. Conclusions: The observed results indicate that the expression levels of HDAC2 become lower and with different subcellular localizations in neurons during hippocampal neuronal maturation, suggesting the specific expression characteristics of HDAC2 might play an important role during postnatal learning-memory function and development.     

生后早期大鼠海马NMDA受体亚单位NR1、NR2A和NR2B的表达变化

运用免疫组织化学反应和图象分析处理技术研究生后 1d、4d、1周、2周、3周、4周、5周、6周的 SD大鼠海马结构中NMDA受体亚单位 NR1、NR2 A、NR2 B三种蛋白质的表达变化规律。结果表明 ,生后各时间点海马结构各区锥体细胞及颗粒细胞胞体中均有 NR1、NR2 A、NR2 B的表达 ,NR2 B还在锥体细胞的顶树突中有较强表达。 NR1与 NR2 B的生后表达变化模式相似 ,在生后 1d和 4d,两者在 CA3区的表达均高于 CA1 区 ;生后 1周后两者在 CA1 区的表达则高于 CA3区 ;到生后 2～ 3周其表达达到峰值。而 NR2 A却与此不同 ,生后 1d、4d时其在海马结构各区的表达较高 ,随发育时间延长表达逐渐下降 ,大约在生后 4周降至谷底。整个发育过程中 ,NR1在海马结构各区的表达始终高于 NR2 A和 NR2 B的表达。这些结果提示 ,生后早期大鼠海马结构 NR1、NR2 A、NR2 B的表达具有发育性时空差异和亚单位差异 ,这种差异可能与发育早期海马学习功能的特异性以及海马各区对缺血敏感性不同有关。     

Alteration of rat hippocampal neurogenesis and neuronal nitric oxide synthase expression upon prenatal exposure to tamoxifen

The present study delineates the effect of tamoxifen on neuronal density and expression of neuronal nitric oxide synthase (nNOS) in hippocampal nerve cells during prenatal and postnatal periods in rats. Pregnant rats were administered with tamoxifen one day prior to labor (E21) and on the childbirth day (E22). Hippocampi of embryos at E22 and newborns at postnatal days of 1, 7, and 21 (P1, P7, and P21) were investigated. Density of the neurons in areas of the developing hippocampus including cornu ammonis (CA1, CA3), dentate gyrus, and subiculum were studied. Our findings show that the number of pyramidal neurons was significantly decreased in CA1 and subiculum of tamoxifen-treated rats in E22, P1, and P7. We found that cellular density was lower in early stages of development, however, cellular density and thickness gradually increased during the development particularly in the third week. We found that nNOS expression was decreased in E22, P1, and P7 in animals treated with tamoxifen. The present study shows that tamoxifen affects development and differentiation of postnatal rat hippocampus, CA1 neurons, and nNOS expression.     

NMDA受体亚单位在海马脑片和在体发育海马结构中表达变化的比较

目的：对比长期培养大鼠海马脑片与在体生后发育海马结构中N-甲基-D-天冬氨酸(NMDA)受体亚单位NR1、NR2A和NR2B表达变化的异同,为利用海马脑片研究NMDA受体亚单位在生理和病理状态中的作用提供资料。方法：免疫组织化学染色、海马脑片培养技术及图像分析。结果：NRI、NR2B在海马脑片各区和生后大鼠海马结构各区表达模式相似,均呈“先升、后降”的趋势,但升降幅度不同;NR2A在海马脑片各区是“先升、后降”的趋势,在生后海马结构各区则呈现“先降后升”的模式。NRI、NR2A、NR2B在脑片和在体发育海马的相似期间,在CA1、CA3、PG区表达均无显著性差异。结论：P3w／C2w时NRI和NR2B在海马脑片各区的表达强度,和其在生后大鼠海马结构中的表达较相似。     

Immunolocalization of NR1, NR2A, and PSD-95 in rat hippocampal subregions during postnatal development

Although the expression of NMDARs and synaptic-associated proteins has been widely studied, the temporospatial distribution of NMDAR subunits and synaptic proteins in different hippocampal subregions during postnatal development still lacks detailed information, and the relationship between NR1 or NR2 subunits and PSD-95 family proteins is controversial. In this study, we used immunofluorescent staining to assess NR1 or NR2A and PSD-95 expressions and the relationship between them in CA1, CA3, and DG of rat hippocampus on postnatal (P) days: P0, P4, P7, P10, P14, P21, P28, P56. The results showed that from P0 to P56, NR1, NR2A, and PSD-95 expressions increased gradually, and the time points of their expression peak differed in CA1, CA3, and DG during postnatal development. Interestingly, although the expression of PSD-95 was positively correlated to both NR1 and NR2A, the NR1 and PSD-95 coexpressed puncta were greatest in CA3, while NR2A and PSD-95 coexpressed puncta were greatest in CA1, compared to other subregions. Surprisingly, at P21, among different strata of CA1, the area of highest expression of NR2A was dramatically changed from stratum pyramidale to stratum polymorphum and stratum moleculare, and returned to stratum pyramidale gradually on the later observed days again, indicating that P21 may be one critical timepoint during postnatal development in CA1. The specific temporospatial distribution pattern of NR1, NR2A, and PSD-95 might be related to the different physiological functions during postnatal development. Discovering the alteration of the relationship between PSD-95 and NMDAR subunits expression may be helpful for understanding mechanisms and therapy of neurodegenerative diseases.     

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.