胚胎脑片免疫荧光组织化学双重漂染技术在神经元发生研究中的应用

目的为更客观地观察胚胎脑神经祖细胞的增殖、分化和迁移,建立胚胎脑片免疫荧光组织化学双重漂染技术。方法灌流固定,取胚胎14天(E14)大鼠脑,低熔点琼脂糖包埋,振动切片机连续冠状切片,免疫荧光组织化学双重漂染,激光扫描共聚焦显微镜下观察。结果波形蛋白(Vimentin)和乙酰胆碱转移酶(ChAT)双阳性细胞呈黄色荧光,胞体位于脑室区,长突起呈放射状。ChAT阳性细胞呈红色荧光,除胞体位于脑室区,长突起呈放射状伸展外,可见皮层板区也有阳性细胞集聚。结论此项技术可直接观察到胚胎脑神经祖细胞和成神经细胞的完整形态,并通过免疫荧光组织化学方法鉴定其表型,从而阐明相互之间的关系。     

原代培养的乙酰胆碱能神经元的表型表达与在体不同（英文）

为了进一步了解原代培养神经元技术是否可以用于建立可靠的乙酰胆碱能神经元体外培养细胞模型,该实验检测了分别培养自E18胎鼠基底前脑(basal forebrain, BF)和海马(hippocampus,HIP)原代培养神经元中的乙酰胆碱能神经元的标志物,同时比较了离体培养细胞与在体在相同脑区中乙酰胆碱能标志物表达的差异。使用免疫标记荧光方法,检测了来自E18胎鼠的基底前脑脑区和海马脑区的离体原代培养神经元中在DIV 3和DIV 21时间点上表达Ch AT和p75NTR(两种常用的胆碱能神经元标记物)的神经元的数量,分析其占总神经元数量的比例,并与E18胎鼠和成年小鼠相同脑区的在体组织切片中的结果相比较。结果显示, Ch AT和p75NTR均在来自基底前脑和海马的培养神经元的DIV 3和DIV 21中高比例表达。然而,虽然在E18胎鼠和成年小鼠的基底前脑的组织切片中有Ch AT和p75NTR的表达,但是在同时期的海马组织切片中并无Ch AT的表达,并且来自基底前脑和海马脑区的培养神经元中表达乙酰胆碱能神经元标志物的神经元数量占总神经元数量比例与在体并不一致。这些结果显示,乙酰胆碱能标志物在离体原代培养和在体中的表达状况可能存在不同。根据实验结果推测,在体外应用原代培养方法培养乙酰胆碱能标志物免疫阳性神经元可能并不是乙酰胆碱能神经元。除了通过免疫组织化学方法,还需要更多的技术和方法来鉴定培养细胞中的乙酰胆碱能神经元。     

他莫昔芬诱导的Cre重组酶在hGfapCreERT2转基因鼠小脑中的表达研究

目的探讨他莫昔芬诱导的hGfapCreERT2转基因鼠小脑中表达Cre重组酶的细胞类型。方法 hGfapCre-ERT2/Rosa26R转基因小鼠在胚胎晚期和出生早期用他莫昔芬诱导Cre重组酶表达,对小脑组织切片行X-gal染色,然后用细胞种类特异性抗体进行免疫组织化学染色,并和X-gal染色双重标记。结果在出生后第7天(P7)、第14天(P14)和第60天(P60),X-gal阳性染色和胶质细胞抗体Blbp阳性染色共标记,和神经元抗体Neun、浦肯野细胞抗体Calbindin及少突胶质细胞前体细胞抗体NG2不共标。结论自胚胎晚期第17.5天(E17.5)后用他莫昔芬诱导hGfapCreERT2转基因鼠,发现Cre重组酶特异性在小脑星形胶质细胞中表达,不在神经元、浦肯野细胞、少突胶质细胞前体细胞中表达。     

大鼠第三脑室室管膜NADPH-d阳性伸展细胞的形态与分布

应用 Ｎ Ａ Ｄ Ｐ Ｈｄ 组织化学方法研究了大鼠第三脑室视前区室管膜的伸展细胞⒚结果表明：１）第三脑室视前区室管膜存在 Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞,其基突伸向视前区并与神经元或毛细血管相接触;２） Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞在第三脑室侧壁常见、室底少见、室顶未发现其存在;３） Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞的形态与分布,在雌、雄大鼠间不存在明显的性别差异⒚尽管 Ｎ Ａ Ｄ Ｐ Ｈｄ 阳性的伸展细胞的生理功能不十分清楚,但本研究为伸展细胞作为脑脑脊液环路的一部分,介导下丘脑对脑脊液中化学变化的感受提供了形态学依据,并提示一氧化氮可能参与了这一过程⒚     

大白鼠第三脑室室管膜的超微结构

本文用扫描和透射电镜证实在成年大白鼠的第三脑室存在室管膜上神经元样细胞、神经胶质细胞和类组织细胞。神经纤维发自神经元样细胞或自脑室外穿入室腔而来,其末梢内含有清亮囊泡或兼有大颗粒囊泡。室腔內尚有膨大的树突末梢和室管膜细胞的球状小体。上述各种结构与感受、分泌和调节功能有关,并为下丘脑控制垂体机能的另一新途径(经脑脊液和室管膜)提供了形态学依据。     

树Qu脑β—内啡肽能神经元胞体和纤维分布的研究

本文用免疫组织化学方法和免疫电镜方法对１４只树Ｑｕβ－内啡肽能神经元胞体和纤维的分布及其在细胞器的定位进行了研究。结果表明,本文首次报道在Ｂｒｏｃａ斜角带观察到β－内啡肽免疫反应阳性神经元胞体,电镜观察到β－内啡肽免疫反应物质定位于大颗粒囊泡内的小颗粒上和粗面内质网上。下丘脑弓状核及其附近区域观察到β－内啡肽免疫反应阳性神经元胞体。在室周区、室旁核、第３脑室室管膜下层及室管膜上皮细胞间、内侧基底下     

大鼠胚胎神经干细胞纹状体内移植后特性

观察大鼠胚胎神经干细胞移植入成年大鼠纹状体后的存活、迁移和分化状况。自14天胎鼠脑室下区分离获得神经干细胞,利用无血清培养基培养扩增并进行鉴定。经4～5代的扩增后,以BrdU标记的神经干细胞通过脑立体定位注射移植入成年大鼠纹状体内,然后分别于移植后2周、4周、6周和8周时做脑冰冻切片,通过免疫组织化学和免疫荧光方法检测移植细胞的数量、定位和分化情况。8周后移植细胞的检出率约16%;移植细胞向周围宿主组织有广泛的迁移表现,尤以沿着白质束向头尾方向的迁移最为显著,最远向后侧达到内囊;纹状体中移植细胞主要分化为神经元和星形胶质细胞。星形胶质细胞数量最多,主要位于移植区与宿主组织临界部位,而神经元处于移植区中央。培养的大鼠胚胎神经干细胞可以作为移植替代治疗神经退行性疾病研究的供体细胞源,而移植中的迁移现象值得注意。     

猫基底前脑胆碱能皮质投射神经元区的P物质样免疫反应神经元

应用免疫组织化学和邻位切片法,研究了猫基底前脑胆碱能皮质投射神经元区的Ｐ物质样免疫反应神经元的分布特征,及其与胆碱乙酰化酶样免疫反应神经元的分布关系,从内侧隔核至Ｍｅｙｎｅｒｔ基底核,２种神经元分布范围相近,且形态,大小相似。但在邻位切片,未见分别呈此二免疫反应的同一神经元的对应剖面。Ｐ物质样免疫反应神经元在内侧隔核和斜角带核数量较多,但在基底核明显减少,在Ｃｈ４间质部及腹侧苍白球连合下部仅为偶见     

大鼠脑中PDGFαR免疫反应阳性细胞与NG2免疫反应阳性细胞的比较

血小板源性生长因子α受体（PDGFαR）是少突胶质细胞前体细胞（OPCs）的特异性标记物,同时另一种膜蛋白NG2也被广泛用于OPCs的研究。为确认PDGFαR免疫反应阳性细胞是否与NG2免疫反应阳性细胞一致。我们运用免疫组织化学、免疫印迹和电生理的方法,检测了新生到老年大鼠脑中PDGFαR和NG2的表达。     

树脑β－内啡肽能神经元胞体和纤维分布的研究

本文用免疫组织化学方法和免疫电镜方法对１４只树脑β－内啡肽能神经元胞体和纤维的分布及其在细胞器的定位进行了研究。结果表明，本文首次报道在Ｂｒｏｃａ斜角带观察到β－内啡肽免疫反应阳性神经元胞体，电镜观察到β－内啡肽免疫反应物质定位于大颗粒囊泡内的小颗粒上和粗面内质网上。下丘脑弓状核及其附近区域观察到β－内啡肽免疫反应阳性神经元胞体。在室周区、室旁核、第３脑室室管膜下层及室管膜上皮细胞间、内侧基底下丘脑及其外侧区、正中隆起内带和外带部可见到β－内啡肽免疫反应阳性纤维和串珠状的膨体。对β－内啡肽的释放途径及其调节因素作了探讨。     

Phosphorylation of 69-kDa choline acetyltransferase at threonine 456 in response to amyloid-beta peptide 1-42

Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons. In the brain, these neurons are especially vulnerable to effects of beta-amyloid (A beta) peptides. Choline acetyltransferase is a substrate for several protein kinases. In the present study, we demonstrate that short term exposure of IMR32 neuroblastoma cells expressing human choline acetyltransferase to A beta-(1-42) changes phosphorylation of the enzyme, resulting in increased activity and alterations in its interaction with other cellular proteins. Using mass spectrometry, we identified threonine 456 as a new phosphorylation site in choline acetyltransferase from A beta-(1-42)-treated cells and in purified recombinant ChAT phosphorylated in vitro by calcium/calmodulin-dependent protein kinase II (CaM kinase II). Whereas phosphorylation of choline acetyltransferase by protein kinase C alone caused a 2-fold increase in enzyme activity, phosphorylation by CaM kinase II alone did not alter enzyme activity. A 3-fold increase in choline acetyltransferase activity was found with coordinate phosphorylation of threonine 456 by CaM kinase II and phosphorylation of serine 440 by protein kinase C. This phosphorylation combination was observed in choline acetyltransferase from A beta-(1-42)-treated cells. Treatment of cells with A beta-(1-42) resulted in two phases of activation of choline acetyltransferase, the first within 30 min and associated with phosphorylation by protein kinase C and the second by 10 h and associated with phosphorylation by both CaM kinase II and protein kinase C. We also show that choline acetyltransferase from A beta-(1-42)-treated cells co-immunoprecipitates with valosin-containing protein, and mutation of threonine 456 to alanine abolished the A beta-(1-42)-induced effects. These studies demonstrate that A beta-(1-42) can acutely regulate the function of choline acetyltransferase, thus potentially altering cholinergic neurotransmission.     

Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS.

Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septal or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.     

Glial cells in coculture can increase the acetylcholinesterase activity in human brain endothelial cells.

The elements of the cholinergic system (acetylcholinesterase and choline acetyltransferase) and butyrylcholinesterase were studied in human cortical capillary samples, brain-derived endothelial cell cultures and glial cell cultures. It was shown that the elements of the cholinergic system are present in the microvessels, but the choline acetyltransferase activity may be due to contamination with cholinergic nerve terminals since no choline acetyltransferase could be demonstrated in endothelial cell cultures. The present results revealed that the activity of acetylcholinesterase is reduced in the cortical endothelial cell cultures after longer culture times, while butyrylcholinesterase activity is not altered. In a system where endothelial cells were cocultured with embryonic human brain astroglial cells for 12 days in vitro, the acetylcholinesterase activity was increased 2-fold. These results support a glial influence on the enzyme activity of the cerebral endothelium.     

Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.     

Interactions Between p75 and TrkA Receptors in Differentiation and Vulnerability of SN56 Cholinergic Cells to Beta-Amyloid

NGF modifies cholinergic neurons through its low-p75 and high affinity-TrkA receptors. Native p75(+)TrkA(–) and trkA-transfected p75(+)TrkA(+) SN56 hybrid cholinergic septal cells were used here to discriminate effects mediated by each receptor. In TrkA(–) cells, NGF (100 ng/ml) affected neither choline acetyltransferase nor morphology but depressed pyruvate dehydrogenase activity by about 30%. Aged 25–35 -amyloid (1 M) caused no changes in choline acetyltransferase and pyruvate dehydrogenase activities in nondifferentiated and differentiated TrkA(–) cells. On the contrary, in nondiferentiated TrkA(+) NGF brought about a 2.5-fold increase of choline acetyltransferase. In differentiated TrkA(+) cells, b-amyloid resulted in no change in PDH but 65% suppression of choline acetyltransferase activity and reduction of their extensions. Thus, activation of TrkA receptors may overcome p75 receptor–mediated inhibitory effects on pyruvate dehydrogenase expression in cholinergic cells. On the other hand, it would make expression of choline acetyltransferase and cell differentiation more susceptible to suppressory effects of -amyloid.     

Cholinergic Deficit Induced by Ethylcholine Aziridinium (AF64A) Transiently Affects Somatostatin and Neuropeptide Y Levels in Rat Brain

The question whether during the process of cholinergic degeneration somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and cortex react to the withdrawal of cholinergic function was addressed. After bilateral intracerebroventricular injection of the cholinotoxin ethylcholine aziridinium (AF64A; 1 or 2 nmol/ventricle) in rats, the activity of choline acetyltransferase (ChAT) started to decline in the hippocampus within 24 h. The reduction of ChAT activity reached its maximum within 4 days (34 and 55% after 1 and 2 nmol of AF64A/ventricle, respectively) and persisted during the observation period of 14 days. In the parietal cortex, ChAT activity decreased by 23% 4 days after 2 nmol of AF64A/ventricle. The loss in ChAT activity was accompanied by a transient decline in the levels of somatostatin and a transient increase in the levels of neuropeptide Y in both brain areas. In the hippocampus, the reduction in somatostatin content was most pronounced after 2 days (by 22 and 33% after 1 and 2 nmol of AF64A/ventricle, respectively). Within 14 days, somatostatin levels returned to control values. Neuropeptide Y levels increased slightly by approximately 25% of control values in the hippocampus. The changes described were present in both the dorsal and ventral subfields of the hippocampus. Similar but less pronounced changes in levels of both neuropeptides were observed in the parietal cortex. The present data provide further evidence for a close neuronal interrelationship between cholinergic and somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and parietal cortex.     

大鼠基底前脑NGF-R及CHAT免疫反应阳性神经元的分布:免疫双标法

本文用免疫组化双标法观察了神经生长因子受体(NGF-R)及胆碱乙酰转移酶(ChAT)免疫反应阳性神经元在成鼠基底前脑内的分布,结果发现嗅结节、隔内侧核、斜角带核、腹侧苍白球及基底大细胞核均有NGF-R及ChAT免疫反应阳性神经元.免疫组化双标染色发现,大部分免疫反应阳性神经元的NGF-R与ChAT共存,部分神经元呈单纯NGF-R或ChAT阳性,但这种NGF-R和ChAT的共存情况在不同区域不完全相同.在隔内侧核和斜角带核,大多数的NGF-R阳性神经元和ChAT阳性神经元共存,但在腹侧仓白球和基底大细胞核,两者共存的神经元较前两区为少.此外ChAT阳性神经元在尾壳核中分布较均匀,而NGF-R阳性神经元较少见.研究结果表明,大多数胆碱能神经元有NGF-R,提示NGF对胆碱能神经元的保护和激活作用,部分可能是通过直接与NGF受体的结合而发生作用.     

Noncorrelation of Choline Kinase or ATP Citrate Lyase with Cholinergic Activity in Rat Brain

Abstract: The activity of choline acetyltransferase was used as an index of cholinergic structures in regions of rat brain. The activities of ATP citrate lyase and choline kinase correlated poorly with cholinergic activity in whole tissue fractions, contrasting with the good correlation between acetylcholinesterase and choline acetyltransferase. Choline acetyltransferase was preferentially localised in synaptosomes prepared from regions of high (striatum) or intermediate (cortex, medulla oblongata/pons) cholinergic activity. In general, this was not true for either choline kinase or ATP citrate lyase.     

The role of extracellular signals in the differentiation of cholinergic neurons from the CNS and PNS in culture

Rat skeletal muscle cells release in culture a macromolecule which stimulates by 25-100 fold the development of choline acetyltransferase (CAT) in cultures of new-born rat sympathetic neurons. This "cholinergic factor" impaired the development of three norepinephrine synthesizing enzymes and of acetylcholinesterase (AChE) in these cultures. The 16S form of AChE failed to develop in cultures grown with the factor, but amounted to 30-40% in 3-week old cultures grown in its absence. Using the development of CAT activity in sympathetic neuron cultures as an assay, the cholinergic factor has been partially purified in 6 steps, and its hydrodynamic parameters determined. The effects of this factor on sympathetic neurotransmitter choice were qualitatively reproduced by 1-10 mM Na butyrate. The cholinergic factor increased CAT activity and decreased AChE in neuron cultures from new-born rat nodose ganglia. The factor also stimulated CAT activity in rat embryo (E14) spinal cord cultures, but stimulated the development of AChE in these cultures.