Functional reconstitution of KCl-evoked,Ca2+-dependent acetylcholine release system in Xenopus oocytes microinjected with presynaptic plasma membranes and synaptic vesicles

We have developed a new method for the generation of functionally active presynaptic chimeras in Xenopus laevis oocytes. Frog oocytes injected with presynaptic subcellular fractions extracted from the electric organ of Torpedo marmorata release acetylcholine in a calcium-dependent manner upon chemical stimulation. Neither oocytes injected without presynaptic plasma membranes nor oocytes injected with ghost erythrocyte plasma membrane instead of presynaptic plasma membrane release acetylcholine. This suggests that specific presynaptic components necessary for KCl-evoked, Ca²⁺-dependent acetylcholine release become functionally integrated in the Xenopus laevis oocytes. Moreover, rhodaminated presynaptic plasma membranes and the synaptic vesicle protein synaptophysin are detected on the oocyte surface by fluorescence or immunofluorescence, respectively, showing that the injected presynaptic components are incorporated into the membrane of the frog oocyte. Furthermore, Botulinum neurotoxin type A, a specific blocker of acetylcholine release in the neuromuscular junction, inhibits the neurotransmitter release from the chimerical oocytes. This suggests that targets for toxin action are also functionally incorporated in the oocyte upon injection of membranous presynaptic components. Our results show that oocytes injected with presynaptic components behave as cholinergic nerve ending chimeras, at least in terms of neurotransmitter release and toxin targets. The system bypasses some problems associated with messenger RNA expression because not only proteins, but native presynaptic components are incorporated. This new technique may provide a useful approach for electrophysiological and pharmacological studies in order to characterize the synaptic transmission. © 1996 Wiley-Liss, Inc.     

大鼠海马CA3区在学习记忆时突触可塑性的变化

目的：探讨学习记忆的形成和遗忘与突触可塑性的关系。方法：用水迷宫训练大鼠21天建立空间辨别性学习记忆模型,随后停止训练,时间分别为3天,7天及14天,用免疫组化方法和图像分析技术,检测了突触素在大鼠海马CA3区表达的变化。结果：对照组大鼠海马CA3区突触素的表达弱,免疫反应产物呈点状颗粒,沿辐射层长轴分布,多形层也有散在分布;模型组大鼠突触素染色明显比对照组的深,密集的小颗粒呈带状沿辐射层排列,在辐射层的底部可见一些大的阳性颗粒,随着停止训练时间的延长,突触素的表达逐渐减弱。结论：空间学习记忆导致海马CA3区辐射层新突触的形成,而随着空间学习记忆的遗忘,又致新形成突触的消退。     

复方瑞康欣胶囊对吗啡依赖戒断大鼠焦虑行为及海马突触素表达的影响

目的 观察复方瑞康欣对吗啡依赖戒断大鼠焦虑行为的影响,并探讨其作用机制.方法 健康雄性SD大鼠,剂量递增法sc吗啡10 d,确认吗啡依赖模型建立成功后,在吗啡自然戒断的1～3 d,复方瑞康欣100、200、300mg/kg和丁螺环酮15 mg/kg ig治疗(2次/d),于末次吗啡注射后72 h,高架十字迷宫实验评价大鼠的焦虑水平,免疫组化检测各组大鼠海马突触素的表达.结果 与生理盐水(NS)治疗组比较,复方瑞康欣200、300 mg/kg和丁螺环酮治疗能明显增加大鼠进入开臂次数和时间的比例(P<0.01、0.05),同时海马突触素的表达显著降低(P<0.01).结论 复方瑞康欣对吗啡依赖大鼠戒断后的焦虑治疗效果明显;逆转焦虑大鼠海马增高的突触素/突触,可能是复方瑞康欣临床治疗吗啡戒断后焦虑的机制之一.     

电针结合重复经颅磁刺激对脑缺血大鼠海马突触素的影响

目的：研究电针结合重复经颅磁刺激对脑缺血大鼠海马突触素的影响。方法：120只Wistar大鼠随机分为正常组、模型组、电针组、重复经颅磁刺激组和电针结合重复经颅磁刺激组, 根据不同时间点每个组又细分为7d、14d和28d组3个亚组。复制大脑中动脉栓塞模型,分别给予电针、磁刺激和电针结合磁刺激干预。免疫荧光标记方法观察缺血侧海马CA3区突触素的表达,蛋白印迹技术定量分析不同时间点、不同组别之间缺血侧海马突触素表达的差异。结果：电针、磁刺激和电针结合磁刺激都能显著上调3个时间点海马突触素的表达。从时间上看,随着治疗时间的增加,突触素的表达逐步上调;不同组间比较,第28天时电针结合磁刺激海马突触素的表达最显著。结论：三种干预方法都能促进脑缺血大鼠海马突触素的表达,其中第28天时电针结合磁刺激效果最显著。     

康复训练对大脑中动脉闭塞大鼠脑梗死灶周围突触素表达的影响

目的:研究康复训练对大鼠脑梗死灶周围突触素(P38)表达的影响。方法:40只Wistar大鼠采用Longa颈外动脉线栓法制备大鼠大脑中动脉闭塞模型,将模型大鼠随机分为康复组、造模对照组,康复组每天进行1小时滚筒、平衡木、转棒及网屏训练,造模对照组置于普通笼中饲养,不予以康复训练。每组随机分为3天、7天、14天、21天4个时间点,每个时间点各5只大鼠,分别于相应天数取脑,采用免疫组织化学方法观察每个时间点脑梗死灶周围突触素的表达。结果:康复组神经功能评分较造模对照组为好(P<0.05);突触素阳性细胞于脑缺血3天后即已出现,7天、14天、21天大量表达,7天为高峰,且康复组较造模对照组多(P<0.05)。随时间延长免疫组化染色变深。结论:康复训练能促进中枢神经系统表达较高水平的突触素。     

骨髓单核细胞移植对脊髓横断大鼠神经营养因子-3和突触素表达的影响

目的:探讨骨髓单核细胞(BMMNCs)移植对脊髓横断(SCT)大鼠脊髓组织中神经营养因子-3(NT-3)和突触素(SYN)表达的影响。方法:146只Winstar大鼠(SPF级,雌雄不限)随机分为3组:SCT组49只,建立SCT模型;实验组49只,建立SCT模型,给予BMMNCs移植治疗;假手术组48只,切除对应的椎板不损伤脊髓;各组大鼠分别于术后5、7、14和21d行后肢功能BBB评分,RT-PCR和免疫组织化学技术检测受损脊髓组织中NT-3和SYN的表达。结果:术后5、7、14和21d各时间点各组大鼠后肢BBB评分比较,SCT组和实验组均低于假手术组(P0.05),且SCT组更低于实验组(P0.05)。术后5、7、14和21d各时间点NT-3阳性细胞数及NT-3mRNA比较,SCT组和实验组均高于假手术组(P0.05),且SCT组更低于实验组(P0.05)。SYN阳性颗粒数及SYN mRNA比较,SCT组和实验组均低于假手术组(P0.05),且SCT组更低于实验组(P0.05)。结论:BMMNCs能够上调局部损伤脊髓NT-3和SYN的表达。     

痴呆模型鼠海马突触素改变及其与学习记忆的关系

探讨痴呆模型鼠海马的突触素改变及其与学习记忆的关系。切断成年SD大鼠左侧穹窿海马伞(Fimbria-Fomix,FF),建立隔海马胆碱能系统损害的痴呆模型。损伤4周后,用Y型迷宫检测大鼠学习记忆能力,免疫组织化学染色和图象分析技术等方法对大鼠海马突触素进行定量分析。结果表明,损伤组学习记忆能力(3.82±0.64)明显低于正常组(8.81±0.32)(P<0.01);与正常组相比,损伤组损伤侧海马CA1区多形层、辐射层、腔隙分子层和齿状回分子层突触素免疫反应物的光密度明显降低,其光密度值分别是43.40±2.57;40.2l±3.22;12.37±1.87;26.63±2.36(P<0.01),并与其学习记忆能力呈显著正相关。本研究结果提示,痴呆模型鼠海马突触素与学习记忆的关系密切。     

当归注射液对全脑缺血后神经再塑影响的实验研究

目的 :研究全脑缺血后海马区的神经再塑及当归注射液的影响。方法 :6 0只健康沙土鼠 ,双侧颈总动脉夹闭 1 0min制作全脑缺血再灌注模型 ,分为治疗组和对照组 ,前者经腹腔给予当归注射液 ,后者给予生理盐水 ,另选 6只沙土鼠作为正常组。于术后 1 6h ,1 ,2 ,3,6d采用免疫组织化学方法检测海马区生长相关蛋白 (growth associ atedprotein ,GAP 4 3)、微管相关蛋白 (microtubule associatedprotein ,MAP 2 )和突触素 (synaptophysin ,SYN)表达的变化。结果 :全脑缺血 1 0min再灌注 1 6h至 6d ,动物脑组织各区尚未见明显形态学变化 ,而MAP 2已随再灌注时间的延长表达水平逐渐降低 ,至再灌注 3d达到最低 ,再灌注 6d又轻度上升 ;GAP 4 3和SYN的表达水平则随再灌注延长而不断上升 ,至再灌注 6d达到最高 ;在各时间点 ,治疗组MAP 2、GAP 4 3和SYN的表达水平均高于对照组 (P <0 .0 5 )。结论 :全脑缺血后海马区有明显的神经再塑和结构重建 ,活血化瘀药当归可加速这一过程     

The prognostic significance of neuroendocrine markers and somatostatin receptor 2 in hepatocellular carcinoma

Prostatic and colon carcinomas with neuroendocrine differentiation are reported to behave more aggressively than those without such differentiation. In hepatocellular carcinomas (HCCs), however, only a few studies have reported the expression status of neuroendocrine markers and somatostatin receptor 2, the main target of a somatostatin analog. Furthermore, the prognostic significance of the markers in HCCs has not been fully explored. We evaluated the expression of the neuroendocrine makers (chromogranin A, synaptophysin, and CD56) and somatostatin receptor 2 (SSTR2) in 95 HCCs, and investigated the correlation between the expression of these markers and clinicopathological findings. Chromogranin A was immunolocalized in 2 cases, synaptophysin in 15 cases, CD56 in 11 cases, and SSTR2 in 19 cases. Immunoreactivity of synaptophysin and CD56 were the significant unfavorable prognostic factors in terms of 2-year disease-free survival (DFS) and the overall survival (OS) along with a high nuclear mitosis level (>10/10 high-power field), a larger tumor size (>5 cm), the presence of vascular and/or biliary invasion, and high TNM stage (III/IV). Multivariate Cox proportional hazards analysis identified synaptophysin as an independent prognostic factor for 2-year DFS and OS. Synaptophysin expression can be used to predict an unfavorable prognosis in patients with HCC.     

Zileuton ameliorates depressive-like behaviors,hippocampal neuroinflammation,apoptosis and synapse dysfunction in mice exposed to chronic mild stress

Our previous study has found that zileuton, a selective 5-lipoxygenase (5LO) inhibitor, abrogated lipopolysaccharide-induced depressive-like behaviors and hippocampal neuroinflammation. Herein, we further extended our curiosity to investigate effects of zileuton on stress-induced depressive-like behaviors. Our data indicated that zileuton significantly ameliorated depressive-like behaviors in mice subjected to chronic mild stress (CMS), as shown in the tail suspension test, forced swimming test and novelty-suppressed feeding test. The further studies indicated that zileuton suppressed hippocampal neuroinflammation, evidenced by lower levels of TNF-α, IL-1β and nuclear NF-κB p65 as well as decreased number of Iba1-positive cells. It also significantly ameliorated hippocampal apoptosis, indicated by deceased number of TUNEL-positive cells, deceased ratio of cleaved caspase-3/procaspase-3 and increased ratio of Bcl-2/Bax. More importantly, zileuton increased the level of synaptic proteins PSD-95 and SYN and the number of NeuN⁺/BrdU⁺ cells in the hippocampus. Over all, zileuton alleviated CMS-induced depressive-like behaviors, neuroinflammatory and apoptotic responses, abnormalities of synapse and neurogenesis in the hippocampus, suggesting that it might has beneficial effects on depression.