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。     

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.     

人参银杏复方制剂对缺氧复氧后大鼠海马CA1区NOS表达的影响

本文旨在观察预防性应用人参银杏复方制剂对缺氧24 h,复氧0、24、72 h时段海马CA1区锥体细胞层神经元Nissl染色变化及海马CA1区一氧化氮合酶(NOS)阳性细胞变化的影响。应用低压氧舱仿海拔8000米高空缺氧模型,以Nissl染色及还原型尼克酰胺腺嘌呤二核苷酸脱氢酶(NADPH-d)组织化学方法并结合图像分析等技术进行研究。结果表明:人参银杏复方制剂能防止缺氧24 h、复氧72 h海马CA1区锥体细胞层神经元的丢失,并能减少缺氧24 h、复氧0、24、72 h时段NOS阳性细胞数量。本研究结果提示:人参银杏复方制剂对缺氧复氧后海马CA1区神经元具有保护作用,其保护作用可能与抑制缺氧复氧后海马CA1区NOS表达有关。     

宫内缺氧对成年小鼠学习记忆能力的影响

目的:探讨孕鼠宫内缺氧对成年小鼠学习记忆能力的影响。方法:用低张性缺氧模型致胎龄13、15、17d小鼠宫内缺氧,新生鼠P1、P7、P14、P28、P90脑组织作Nissl染色,观察海马CA3区锥体细胞层神经元数目及形态,P90小鼠行Morris水迷宫实验。结果:与正常组比较,宫内缺氧组小鼠海马CA3区锥体细胞层神经元数量明显减少,逃避潜伏期延长及穿环次数明显减少。结论:宫内缺氧导致小鼠海马CA3区锥体细胞层神经元数目减少及成年小鼠学习记忆能力降低。     

P>四氯化碳对大鼠海马CA1区神经元低亲和力神经生长因子受体p75表达的影响/P>

目的 研究四氯化碳(CCl4)对大鼠脑海马CA1区神经元低亲和力神经生长因子受体p75的表达和凋亡的影响.方法 将24只健康雄性Wistar大鼠随机分为对照组和CCl4组.对照组每周一、四背部皮下注射橄榄油(0.12ml/100g),CCl4组分别在每周一或/和周四背部皮下注射60?l4-橄榄油溶液(0.3ml/100g)1次(1d组)、2次(1周组)和4次(2周组),实验结束时处死大鼠.取脑后沿正中矢状面切开,右侧半脑石蜡切片,行Nissl染色,观察海马CA1区神经元的形态学改变;行p75、Bax免疫组织化学染色和Western blotting分析,检测海马CA1区神经元p75、Bax的表达,行原位缺口末端标记法(TUNEL)观察海马CA1区神经元的凋亡.结果 Nissl染色显示对照组CA1区神经元染色较深,细胞数目较多,排列整齐,神经元内可见大量尼氏体;CCl4组CA1区神经元排列较对照组紊乱,部分锥体细胞体积缩小,核固缩,呈三角形,胞浆内尼氏体数目减少或消失.对照组海马CA1区可见少量表达p75和Bax的阳性细胞;CCl4组海马CA1区表达p75和Bax的阳性细胞数较对照组明显增多,以1周CCl4组增多最为明显;Western blotting结果与免疫组织化学染色结果一致.对照组海马CA1区未见TUNEL阳性细胞;CCl4组海马CA1区可见大量细胞核呈棕黄色着色的TUNEL阳性细胞,以1周CCl4组的阳性细胞数最多.结论 注射CCl4后,大鼠脑海马CA1区p75的表达明显增强,Bax的表达也相应增强,凋亡的神经元明显增多.     

人胎儿海马发育的初步观察

取4—10个月的人胎儿海马中段,作沿长轴横切片,Nissl法染色后,观察其发育过程中锥体细胞最的锥体细胞和齿状回的颗粒细胞的变化。结果发现随着海马的发育,细胞的密度变小,体积变大,并且这种变化有快慢交替的情况。     

氯化锂匹罗卡品诱导癫痫大鼠海马神经元GPER1表达的变化

目的:观察G蛋白偶联雌激素受体1(GPER1)在癫痫大鼠海马神经元中表达的变化。方法:成年雄性SD大鼠分成对照组(control)和癫痫组(epilepsy),利用腹腔注射氯化锂-匹罗卡品方法制备癫痫模型,分别在1、2、3、7、14 d和28 d,利用Morris水迷宫检测大鼠学习记忆能力,利用尼氏染色观察大鼠海马神经元形态变化;利用免疫组化和Western Blot技术观察GPER1在海马的表达。结果:水迷宫结果显示,与Control组相比,造模14 d的大鼠逃逸潜伏时间明显延长(P 0. 05),穿越目标象限区域的次数较Control组显著降低(P 0. 05)。尼氏染色结果显示:与Control组相比,造模1 d和2 d的大鼠CA1及CA3区锥体细胞层细胞及DG区颗粒细胞层细胞体积缩小,细胞间距增加,尼氏染色减弱;造模3和7 d的大鼠细胞体积明显缩小,细胞间隙明显增大,尼氏染色加深,CA1及CA3细胞数量明显减少;造模14 d和28 d的大鼠神经元体积逐渐向正常恢复,但仍较Control组小。免疫组化结果显示:GPER1免疫阳性细胞以海马锥体细胞和齿状回颗粒细胞为主,主要分布在细胞膜。与Control组相比,造模2 d和3 d的大鼠海马CA1及CA3区GPER1表达增加(P 0. 05),7 d后增加最明显(P 0. 01),14、28 d后表达下降;在DG区,造模3 d及7 d的大鼠GPER1表达增加(P 0. 05),14 d后表达下降(P 0. 05),28 d大鼠无显著差异。Western Blot结果显示:与Control组比较,造模2 d和3 d的大鼠GPER1相对表达量开始增高,7 d后明显增高(P 0. 05),14 d及28 d的大鼠表达降低。结论:GPER1在海马神经元的表达随着神经元损伤的加重而增高,随着神经元损伤的恢复逐渐降低,提示其表达变化与神经元的损伤与修复有关。     

巢蛋白在成年大鼠海马神经元的表达及巢蛋白阳性神经元的生后发育

本文采用免疫组织化学方法对成年大鼠海马巢蛋白阳性神经元及其出生后的发育变化进行了研究。结果显示 ,在成年大鼠海马 CA1、CA2和 CA3区锥体层某些特定部位的神经元呈巢蛋白阳性反应。巢蛋白阳性神经元出生后 3周开始在海马中出现 ,并且到 12月龄大鼠海马内仍有存在。双重反应结果显示 ,巢蛋白与 GABA在海马神经元内无共存。成年大鼠海马锥体层神经元表达巢蛋白的生物学意义尚不清楚     

NR2B反义寡核苷酸对大鼠海马CA1区NR2B mRNA表达的影响

目的：观察NMDA受体2B亚单位（NR2B）反义寡核苷酸（ANR2B）对海马CA1区NR2B mRNA表达的影响,筛选有效的ANR2B,探讨改变NR2B mRNA表达的新方法.方法：正常SD大鼠,海马CA1区立体定位注射ANR2B,灌注取脑,连续冰冻切片,原位杂交组织化学方法染色,光镜下观察NR2B mRNA在ANB2B作用下的表达变化.结果：正常大鼠海马CA1区立体定位注射ANR2B后,注射点及其周围NR2B mRNA原位杂交染色强度明显下降,仅有少量锥体细胞和颗粒细胞散在分布;而在注射NR2B正义寡核苷酸（SNR2B）、生理盐水（NS）及NS插针不注射（NSNO）的海马切片上,海马CA1区的染色特征与注射ANR2B有明显差别,其作用区锥体细胞、颗粒细胞及顶树突的NR2B原位杂交染色强度无明显变化.结论：ANR2B能够降低NR2B mRNA在正常大鼠海马CA1区的基础性高表达.     

突触结合蛋白I和Ⅳ在小鼠背海马表达的区域差异及年龄相关性变化

目的:探讨昆明小鼠背海马突触前囊泡蛋白(Syt)Ⅰ和Ⅳ的分布差异及其年龄相关性变化.方法:选取3个年龄段昆明小鼠22月龄17只、11月龄22只和6月龄28只作为研究对象,采用免疫组织化学技术检测其背海马Syt Ⅰ和Syt Ⅳ含量.结果:SytⅠ与Syt Ⅳ在不同年龄组小鼠背海马锥体细胞和颗粒细胞中均有表达,其中SytⅠ在CA3区透明层和齿状回(DG)门区呈高表达,而Syt Ⅳ在锥体细胞和颗粒细胞胞膜呈高表达.22月龄鼠在海马CA1、 CA3和DG中Syt Ⅰ的相对含量高于11月龄和6月龄鼠,而Syt Ⅳ的相对含量低于6月龄鼠.结论:相对于中、青年鼠而言,老年昆明小鼠背海马Syt Ⅰ水平升高而Syt Ⅳ水平降低;Syt Ⅰ的优势表达可能在苔藓纤维及其与CA3区锥体细胞顶树突形成的轴-树或树-树型突触,Syt Ⅳ的优势表达可能在锥体细胞和颗粒细胞的轴-体或树-体型突触.     

Newborn granule cells in the ageing dentate gyrus

The dentate gyrus of the hippocampus generates neurons throughout life, but adult neurogenesis exhibits a marked age-dependent decline. Although the decrease in the rate of neurogenesis has been extensively documented in the ageing hippocampus, the specific characteristics of dentate granule cells born in such a continuously changing environment have received little attention. We have used retroviral labelling of neural progenitor cells of the adult mouse dentate gyrus to study morphological properties of neurons born at different ages. Dendritic spine density was measured to estimate glutamatergic afferent connectivity. Fully mature neurons born at the age of 2 months display ∼2.3 spines μm⁻¹ and maintain their overall morphology and spine density in 1-year-old mice. Surprisingly, granule cells born in 10-month-old mice, at which time the rate of neurogenesis has decreased by ∼40-fold, reach a density of dendritic spines similar to that of neurons born in young adulthood. Therefore, in spite of the sharp decline in cell proliferation, differentiation and overall neuronal number, the ageing hippocampus presents a suitable environment for new surviving neurons to reach a high level of complexity, comparable to that of all other dentate granule cells.     

Neurons born in the adult dentate gyrus form functional synapses with target cells

Adult neurogenesis occurs in the hippocampus and the olfactory bulb of the mammalian CNS. Recent studies have demonstrated that newborn granule cells of the adult hippocampus are postsynaptic targets of excitatory and inhibitory neurons, but evidence of synapse formation by the axons of these cells is still lacking. By combining retroviral expression of green fluorescent protein in adult-born neurons of the mouse dentate gyrus with immuno-electron microscopy, we found output synapses that were formed by labeled terminals on appropriate target cells in the CA3 area and the hilus. Furthermore, retroviral expression of channelrhodopsin-2 allowed us to light-stimulate newborn granule cells and identify postsynaptic target neurons by whole-cell recordings in acute slices. Our structural and functional evidence indicates that axons of adult-born granule cells establish synapses with hilar interneurons, mossy cells and CA3 pyramidal cells and release glutamate as their main neurotransmitter.     

Localization of copper-zinc superoxide dismutase mRNA in human hippocampus by in situ hybridization

The cellular localization of copper-zinc superoxide dismutase (CuZn SOD) mRNA was determined in the human hippocampus by in situ hybridization with a 35S-labelled DNA probe complementary to human CuZn SOD mRNA. A positive hybridization signal was detected in pyramidal cell layers CA1-CA4 of Ammon's horn (CA), pyramidal cells of subiculum and in the granule cells of the dentate gyrus. The fact that CuZn SOD gene expression is important in neurones which are preferentially vulnerable in neurodegenerative processes such as Alzheimer's disease, suggests a role played by oxygen free radicals in the mechanism of nerve cell death.     

Development of full-length Trk B-immunoreactive structures in the hippocampal formation of the macaque monkey

Distribution and morphological changes of cells containing the signal transducing neurotrophin receptor, full-length Trk B (fl-Trk B), were investigated in the hippocampal formation of the macaque monkey between embryonic day 140 and the adult stage. Western blot analysis showed that one main protein band, which migrated at 141 kDa, was detected in both the embryonic and adult hippocampal formation. In the pyramidal cells in CA1 and CA3 subfields, the subiculum, and the entorhinal cortex, fl-Trk B-immunoreactive dendrites were observable in the embryonic stage. In contrast, in the granule cells of the dentate gyrus, few dendrites were immunoreactive during embryonic and early developmental stages. This difference may be due to the later growth of the granule cells of the dentate gyrus. The existence of fl-Trk B immunoreactivity in the cell body and dendrites in the embryonic hippocampal neurons, suggests that BDNF and/or NT4/5 act on the hippocampal cells by autocrine/paracrine mechanisms. In the entorhinal cortex, fl-Trk B immunoreactivity became localized in the stellate cells in layer II and the pyramidal cells in layers III, V and VI in adulthood. This indicates that BDNF and/or NT4/5 are important for the maintenance of the projection neurons in the entorhinal cortex at the adult stage. The strongest fl-Trk B immunoreactivity in the hippocampal neurons occurred at postnatal month 4, corresponding to the period of greatest synapse production in the monkey hippocampus, suggesting that BDNF and/or NT4/5 with fl-Trk B may play a role in synapse formation in the monkey hippocampus.     

Short-term glucocorticoid manipulations affect neuronal morphology and survival in the adult dentate gyrus

In order to determine whether short-term glucocorticoid manipulations influence the morphology and survival of neurons in the adult mammalian hippocampal formation, we performed quantitative analyses of Golgi-impregnated and Nissl-stained tissue from the brains of sham operated male rats, adrenalectomized male rats and adrenalectomized male rats which received corticosterone replacement. Three days after adrenalectomy, massive cell death, as detected by a dramatic increase in number of pyknotic cells, was observed in the granule cell layer of the dentate gyrus. By seven days following adrenalectomy, the numbers of pyknotic cells were even greater. Moreover, significant decreases in cross-sectional cell body area and numbers of dendritic branch points of Golgi-impregnated dentate gyrus granule cells were detected at seven days after adrenalectomy. Replacement of corticosterone to adrenalectomized rats prevented the appearance of large numbers of pyknotic cells as well as the decrease in granule cell cross-sectional cell body area and the numbers of dendritic branch points. In contrast, no obvious signs of degeneration were detected in the pyramidal cell layers of the CA1 and CA3 regions of the hippocampus at either three or seven days following adrenalectomy. In addition, no significant changes in morphological characteristics were observed in CA1 or CA3 pyramidal cells with adrenalectomy. These results show that dentate gyrus granule cells require glucocorticoids for their survival and for the maintenance of normal morphology and suggest that granule cell morphology and/or survival may undergo constant fluctuation in response to diurnal rhythms or stress-induced changes in glucocorticoid levels.     

Chronic non-invasive glucocorticoid administration decreases polysialylated neural cell adhesion molecule expression in the adult rat dentate gyrus

The expression of the polysialylated neural cell adhesion molecule (PSA-NCAM) is increased in the hippocampus after chronic restraint stress (CRS) and may play a permissive role in structural changes that include dendrite reorganization in dentate gyrus (DG) and CA3 pyramidal neurons and suppression of neurogenesis in DG. We report that chronic oral corticosterone (CORT) administration decreases the number of PSA-NCAM immunoreactive granule neurons in the adult rat dentate gyrus, and the available evidence suggests that this is an indirect effect of CORT, possibly involving excitatory amino acids, that may not be directly related to neurogenesis. Because CORT treatment reduces but does not eliminate PSA-NCAM expression, the present results do not exclude a permissive role for PSA-NCAM in CORT or CRS-induced structural plasticity in hippocampus.     

Cloning and characterization of guanine deaminase from mouse and rat brain.

A search for genes differentially expressed in the rat striatum revealed a gene fragment with a ventral to dorsal striatal expression pattern. The sequence of the fragment was used to isolate mouse and rat clones that upon sequencing were identified as homologous to human guanine deaminase. Here we report the distribution of guanine deaminase in the rodent brain. In situ hybridization localization of the encoding mRNA showed a distribution primarily in forebrain areas including cortical pyramidal neurons, ventral striatal medium spiny neurons, hippocampal pyramidal neurons in CA3-CA1 and granule cells in the dentate gyrus, and neurons of the amygdala. Immunohistochemistry using antibodies raised against peptide fragments derived from the guanine deaminase protein sequence showed localization of guanine deaminase in areas predicted by the mRNA distribution. In addition to immunolabeling of neurons in the cerebral cortex, hippocampus, striatum and amygdala there was also labeling in the terminal fields of these neurons including the thalamus, globus pallidum and substantia nigra. A functional histochemical assay that demonstrates the site of guanine deamination shows guanine deaminase activity in a pattern that matched the immunohistochemical localization. The cellular distribution of guanine deaminase to distal areas of the cell including terminals and dendrites was additionally demonstrated by the expression of recombinant guanine deaminase in transformed cortical neurons in culture.In summary we have described the isolation and characterization of mouse and rat guanine deaminase. The expression of guanine deaminase is primarily restricted to forebrain neurons. A histochemical assay was used to localize guanine deaminase activity to the dendrites and axons of neurons expressing guanine deaminase.     

Septal cholinergic afferents regulate expression of brain-derived neurotrophic factor andβ-nerve growth factor mRNA in rat hippocampus

Summary In situ hybridization was used to study the expression of members of the nerve growth factor family of trophic factors in rat hippocampus following stimulation of afferent cholinergic and glutamatergic pathways with quisqualate. A transient increase in brain-derived neurotrophic factor (BDNF) and-nerve growth factor (NGF) mRNA expression in the hippocampus was seen 4 h after a quisqualate injection into the medial septal nucleus. Both BDNF and NGF mRNA levels increased more than 4-fold in the granule layer of the dentate gyrus and for BDNF mRNA also in the pyramidal cells of CA1, while the levels of BDNF mRNA in CA3 increased 2-fold. The increase in BDNF and NGF mRNA levels were completely prevented by pretreatment with systemic injections of either scopolamine or diazepam. A quisqualate injection into the entorhinal cortex, containing glutamatergic afferents to the hippocampus, resulted in a 15-, 5- and 17-fold increase in the expression of BDNF mRNA in the ipsilateral granule cells, CA3 and CA1 pyramidal cells, respectively. Similar increases were also seen in the hippocampus contralateral to the injections. In contrast, the level of NGF mRNA did not increase significantly in any of the subfields in the hippocampus. The increase in BDNF mRNA after cortex injections was attenuated by diazepam but not by scopolamine. These findings imply that increased activity in afferent cholinergic and glutamatergic pathways to the hippocampus differentially regulate expression of the NGF family of neurotrophic factors in the hippocampus.     

Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain

Neurogenesis occurs continuously in the forebrain of adult mammals, but the functional importance of adult neurogenesis is still unclear. Here, using a genetic labeling method in adult mice, we found that continuous neurogenesis results in the replacement of the majority of granule neurons in the olfactory bulb and a substantial addition of granule neurons to the hippocampal dentate gyrus. Genetic ablation of newly formed neurons in adult mice led to a gradual decrease in the number of granule cells in the olfactory bulb, inhibition of increases in the granule cell number in the dentate gyrus and impairment of behaviors in contextual and spatial memory, which are known to depend on hippocampus. These results suggest that continuous neurogenesis is required for the maintenance and reorganization of the whole interneuron system in the olfactory bulb, the modulation and refinement of the existing neuronal circuits in the dentate gyrus and the normal behaviors involved in hippocampal-dependent memory.