Telemetric recordings of single neuron activity and visual scenes in monkeys walking in an open field

This paper describes a portable recording system and methods for obtaining chronic recordings of single units and tracking rhesus monkey behavior in an open field. The integrated system consists of four major components: (1) microelectrode assembly; (2) h

Reinforcement-related neurons in the primate basal forebrain respond to the learned significance of task events rather than to the hedonic attributes of reward

The objective of this study was to determine if the responses of basal forebrain neurons are related to the cognitive processes necessary for the performance of behavioural tasks, or to the hedonic attributes of the reinforcers delivered to the monkey as

CORRELATION OF ZONA-BINDING WITH OOCYTE MATURATION AND SPERM MOTILITY IN RHESUS-MONKEYS BY HEMIZONA ASSAY

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PB1). The mean numbers of sperm bound to hemizona for PBI, PVS, GV, and MC groups were 132.9 +/- 12.0, 71.5 +/- 10.1, 36.1 +/- 4.0, and 20.1 +/- 2.9 (Mean +/- SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 +/- 2.2 and 25.3 +/- 2.9 (Mean +/- SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1)The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the ''zona maturation'' hypothesis. (C) 1994 Wiley-Liss, Inc.

肝细胞生长因子促进猕猴胚胎干细胞来源的神经前体细胞的增殖

肝细胞生长因子（HGF）是一个多效应因子，在神经系统中具有重要作用， 但是对于HGF在早期神经系统发育（特别是哺乳动物）中的具体作用还不明确， 这方面的研究还很少。我们早前的研究发现采用HGF和G5 supplement结合EB法 可诱导猕猴胚胎干细胞（rESCs）定向分化成高纯度（88.3± 8.1%）的可移植的 神经前体细胞，但是HGF在整个分化过程中的具体作用及HGF与其它因子的关 系还不清楚。 本研究改进先前研究体系，用单层培养诱导体系代替EB法诱导体系，用bFGF 代替G5 supplement。即采用单层培养的方式，分别用同时含bFGF和HGF、只含 HGF或bFGF以及两者都没有添加的成分确定的分化液诱导rESCs向神经细胞分 化。并检测HGF对rESCs来源的神经前体细胞的增殖速度的影响，旨在进一步研 究HGF在整个分化过程中的具体作用及HGF与bFGF的关系。主要结论如下：1） 采用单层培养法，同时添加HGF和bFGF可诱导rESCs在两周内定向分化为高纯度 （>85%）的神经前体细胞，从而建立了一种更为简单的诱导rESCs分化成神经细 胞的方法；2）不同分化条件下都得到了相似比例的神经前体细胞，表明外源性 的HGF在诱导rESC向神经前体细胞转变的过程中对于神经细胞命运的决定并不 起作用；3）HGF能有效地促进rESCs来源的神经前体细胞的增殖，并且与bFGF 具有协同作用。

Comparative Analysis of Five Different Homologous Feeder Cell Lines in the Ability to Support Rhesus Embryonic Stem Cells

我们以前的研究建立了五株猕猴饲养层细胞系来支持猕猴胚胎干细胞(rESCs)的生长:一岁猴耳皮肤成纤维细胞(MESFs)、两岁猴输卵管成纤维细胞(MOFs)、成年猴卵泡颗粒成纤维样细胞(MFGs)、成年猴卵泡颗粒上皮样细胞(MFGEs),以及MESFs的克隆成纤维细胞(CMESFs).我们发现MESFs、CMESFs、MOFs和MFGs,而不足MFGEs支持猕猴胚胎干细胞(rESCs,rhesus embryonic stem cells)的生长.通过半定量PCR的方法,我们在支持性的饲养层细胞中检测到了一些基因的高表达.在本研究中,我们运用Affymetrix公司的GeneChip Rhesus Macaque Genome Array芯片来研究这五株同源饲养层的表达谱,希望发现哪些细胞因子和信号通路在维持rESCs中起到重要作用.结果表明,除MFGE外,包括GREM2、bFGF,、KITLG,、DKK3、GREM1、AREG、SERPINF1和LTBF1等八个基因的mRNA在支持性的饲养层细胞中高表达.本研究结果提示,很多信号通路在支持rESCs的未分化生长和多潜能性方面可能起到了冗余的作用.

体外分步诱导猕猴胚胎干细胞为肝细胞

采用分阶段诱导方法模拟肝细胞体内发育,建立体外诱导猕猴胚胎干细胞(rhesus monkey embryonic stem cells,rESCs)分化为成熟肝细胞的体系,对研究以ES细胞为基础的临床替代治疗人类晚期肝脏疾病具有重要的意义.将rESCs团块在含有10%FBS的DMEM培养基中悬浮培养11d,形成含有早期内胚层细胞的拟胚体(embryonic bodies,EB)并开始表达早期肝细胞的部分基因或蛋白,将11日龄EB接种至包被有ECM的组织培养皿,分阶段加入aFGF、BMP-4及OSM.经aFGF和BMP-4诱导7～10d后,分化细胞形态变为具有双核的多角形细胞,表达早期和中期肝细胞特异性的蛋白(AFP、ALB及CK18)和基因(AFP、ALB、APOH,G-6-P及TAT),并具有储存糖原的功能.撤除aFGF和BMP-4,添加OSM继续诱导7～10 d,分化的细胞表达成熟肝细胞所特有基因CYP1B1和ADH1C,并具有摄取靛青绿的能力.

肝细胞生长因子促进猕猴胚胎干细胞来源的神经前体细胞的增殖

肝细胞牛长因子(hepatoeyte growth factor,HGF)足一个多效应因子,在神经系统中具有重要作用.早前的研究发现采用HGF和G5 supplement结合EB(embryoid body)法可诱导猕猴胍胎干细胞(rhesus embryonic stem cells,rESCs)定向分化成高纯度的可移植的神经前体细胞(neural progenitors),但对于HGF在整个诱导分化过程中的具体作用及机制还不清楚.本研究改进先前研究体系,采用单层培养法,同时添加HGF和bFGF(basic fibroblast growth factor,碱性成纤维细胞生长因子)诱导rESCs在两周内定向分化为高纯度[(81.66±4.37)%]的神经前体细胞,并且单独添加HGF或bFGF以及两者都没有添加的条件下也得到了相似比例的神经前体细胞,表明外源性的HGF在诱导rESCs向神经前体细胞转变的过程中对十神经细胞命运的决定并不起作用;进一步研究发现HGF能有效地促进神经前体细胞的增殖,并且与bFGF具有协同作用.总之,本研究建立了一种更为简单的诱导rESCs分化成神经细胞的方法,发现外源性的HGF在rESCs向神经前体细胞分化的过程中并没有神经诱导的作用,但能与bFGF协同作用促进rESCs来源的神经前体细胞的增殖.

饲养层FGF2表达量对猕猴胚胎干细胞自我更新及多能性的影响

用转基因和RNA干扰(RNAi)法建立5组不同成纤维细胞生长因子-2(fibroblast growth factor-2,FGF2)表达量的猕猴耳部皮肤成纤维细胞(MESF)系:过表达FGF2组(f1),过表达的阴性对照组(f2),FGF2 RNA干扰组(f3),RNA干扰的阴性对照组(f4)和未作任何处理的对照组(f5).5组MESF的FGF2表达量相对值为f1:f2:f3:f4:f5=4:2:1:2:2;c-fos,TGF-β1,INHBA,Gremlinl在f1中表达量上升,在f3中表达量下降;BMP4,TGF-β2在f1中表达量下降,在f3中表达量上升;表明内源FGF2能够作用于MESF的TGF-β信号通路,引起相关基因表达量的变化.用这砦细胞作为饲养层长期培养(10代)猕猴胚胎干细胞(RhESC),结果在f1上培养的RhESC增殖速度都比对照组快,并且c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达量均上升,BMP4表达下调;在f3上培养的RhESC增殖较慢,BMP4表达上调,c-fos,TGF-β1,INHBA,Gremlinl,Oct-4,Nanog,Sox2表达下调.5组MESF上培养的RhESC形成的EB均表达各胚层早期标记基因(marker),说明RhESC的多能性没有受到影响,但表达量有差异,f1上RhESC形成的EB所有marker都低表达.结果表明饲养层的FGF2含量不仅影响自身相关基凶的表达,还对RhESC的自我更新有一定的作用.

兔抗人外周血单核细胞、兔抗猴脾脏淋巴细胞免疫血清的制备及其在分离人和猕猴胚胎内细胞团中的应用

采用梯度离心的方法用淋巴细胞分离液从浓缩的人白细胞悬液中分离人外周血单核细胞，从猕猴新 鲜脾脏中研磨分离猕猴脾脏淋巴细胞；分别将人外周血单核细胞和猕猴脾脏淋巴细胞作为免疫原，每次通过耳 缘静脉注射4×10s个细胞免疫日本大耳白兔，每周免疫一次，共免疫3次，制备兔抗人外周血单核细胞和兔抗 猕猴脾脏淋巴细胞免疫血清。体外培养人和猕猴胚胎至囊胚，采用制备的免疫血清，分离人和猕猴囊胚内细胞 团，用于建立人和猕猴胚胎干细胞系。结果如下：(1)用半微量细胞毒实验法测得兔抗人外周血单核细胞免疫 血清和兔抗猕猴脾脏淋巴细胞免疫血清的效价分别为1：320和1：640；(2)两种免疫血清成功地裂解了15个人 囊胚和33个猕猴囊胚的滋养层细胞，分离出了内细胞团，表明免疫血清的制备取得了成功，为建立人和猕猴 胚胎干细胞系奠定了基础。

不同渗透压的稀释液对猕猴精子低温冷冻保存的影响

以稀释液TTE (382 mOsm/ kg) 为对照, 研究了5 种渗透压(688 、389 、329 、166 、43 mOsm/ kg) 的TEST 稀释液(TEST、mTEST1、mTEST2、mTEST3、mTEST4) 在冷冻过程中对猕猴精子功能的影响。精液一 步稀释于含甘油的防冻液中, 甘油的终浓度为5 % (v/ v) 。在冷冻前后分别检测精子的运动度和质膜完整性, 后者用Hoechst 33342 和碘化丙锭双色标记流式细胞术分析。结果表明: 冷冻之前, 与鲜精相比, 用TEST 和 mTEST4 稀释的精子运动度和质膜完整性显著降低( P < 01001) , 其余组中除mTEST2 稀释的精子质膜完整性显 著降低( P < 0105) 外, 精子运动度无差异; 冷冻复苏后, TTE、mTEST3 和mTEST1 冻存精子的运动度和质膜 完整性最高, 其次是mTEST2 , TEST和mTEST4 冷冻效果最差( P < 0105) 。提示等渗、适当高渗或低渗的稀释 液适合猕猴精子的冷冻保存; 对精子产生高渗毒害作用是导致猕猴精子用TEST 冷冻存活率低的主要原因。

四种渗透性防冻剂在猕猴精子低温冷冻保存中对精子功能状态的影响

以冷冻精子的复苏运动度、荧光染料Hoechst 33258 检测的细胞膜完整率、异硫氰酸荧光素标记的 花生凝集素(FITC2PNA) 检测的顶体完整率作为精子功能状态的指标, 对甘油、二甲亚砜、乙二醇和丙二醇4 种常用渗透性防冻剂在猕猴精子冷冻保存过程中的作用进行了比较。结果表明: 冷冻保存精子的复苏运动度, 甘油(4713 ±517 %) 和乙二醇(4418 ±617 %) > 二甲亚砜(2219 ±019 %) > 丙二醇(0 ±0 %) ; 细胞膜完整 率, 甘油(5418 ±312 %) 和乙二醇(5410 ±617 %) > 二甲亚砜(3715 ±710 %) > 丙二醇(2813 ±615 %) ; 顶 体完整率, 甘油(8212 ±214 %) 和乙二醇(8214 ±214 %) > 二甲亚砜(6817 ±517 %) 和丙二醇(7213 ± 315 %) ( P < 0105) 。结果提示: 二甲亚砜和丙二醇, 尤其是丙二醇并不适合猕猴精子的冷冻保存; 而乙二醇具 有和甘油相似的保护作用, 是一种极具潜力的猕猴精子冷冻保存的渗透性防冻剂。

DRAMATIC CONSEQUENCES OF FOOD HANDOUTS TO MACACA-THIBETANA AT MOUNT-EMEI, CHINA

Food handouts to Macaca thibetana at Mt. Emei have had dramatic consequences for both man and monkey as tourism has increased over the last decade. Food handouts and human submissive behaviour facilitate beg-robbing by the monkeys. which can be regarded as a mixed conditioning chain. Because of their lack of understanding of primate behaviour and resulting inappropriate responses, many visitors have lost possessions and have been severely frightened or even injured; in fact there have been 1 0 deaths as an indirect result over that past 8 years. The appropriate human response proved to be the display of dominance to maintain a distance from a beg-robbing monkey. Road-ranging macaques have also been injured or killed by visitors to obtain meat or bones or merely for amusement. Attempts should be made to eliminate the negative effects of food handouts by increasing visitors' awareness of behavioural and ecological aspects and through aversive conditioning of the macaques.

DOPAMINE D2 RECEPTOR MECHANISMS CONTRIBUTE TO AGE-RELATED COGNITIVE DECLINE - THE EFFECTS OF QUINPIROLE ON MEMORY AND MOTOR-PERFORMANCE IN MONKEYS

The D2 dopamine (DA) receptor agonist, quinpirole, was characterized in young adult monkeys, young reserpine-treated monkeys and aged monkeys to assess the contribution of DA to age-related loss of prefrontal cortical (PFC) cognitive function, Monkeys were tested on a delayed response memory task that depends on the PFC, and a fine motor task that taps the functions of the motor cortex, In young adult monkeys, low quinpirole doses impaired performance of the PFC and fine motor tasks, while higher doses improved memory performance and induced dyskinesias and ''hallucinatory-like'' behaviors. The pattern of the quinpirole response in reserpine-treated monkeys suggested that the impairments in delayed response and fine motor performance resulted from drug actions at D2 autoreceptors, while the improvement in delayed response performance, dyskinesias and ''hallucinatory-like'' behaviors resulted from actions at postsynaptic receptors. In aged monkeys, low doses of quinpirole continued to impair fine motor performance, but lost their ability to impair delayed response performance. The magnitude of cognitive improvement and the incidence of ''hallucinatory-like'' behaviors were also reduced in the aged animals, suggesting some loss of postsynaptic D2 receptor function, The pattern of results is consistent with the greater loss of DA from the PFC than from motor areas in aged monkey brain (Goldman-Rakic and Brown, 1981; Wenk et al., 1989), and indicates that DA depletion contributes significantly to age-related cognitive decline.

COMPARISON OF MASTICATORY MORPHOLOGY BETWEEN RHINOPITHECUS-BIETI AND R-ROXELLANA

The masticatory apparatus for two endemic species of golden monkey in China, Rhinopithecus bieti and Rhinopithecus roxellana, were compared with those of macaques, Macaca and leaf monkeys, Presbytis. Multivariate analyses demonstrated that the two golden monkey species are distinct. Interspecies allometric analyses revealed that golden monkeys differ in their masticatory apparatus from both macaques and leaf monkeys. The prominent symphysial fusion, corpus, and sagittal condylar dimension of R. roxellana may produce efficient biting force on the incisal and posterior canine teeth, with the heavy reaction force barn on the temporomandibular joint. However, the well-developed bizygamatic width and mandibular height in R. bieti suggest that posterior canine function is similarly prominent in R. roxellana, while incisal function is not. (C) 1995 Wiley-Liss, Inc.

Habitat degradation of Rhinopithecus bieti in Yunnan, China

Black-and-white snub-nosed monkeys (Rhinopithecus bieti) are endemic to the Trans-Himalayas in Northwest Yunnan and Southeast Tibet between the upper Yangtze and Mekong Rivers. Based on field surveys and previous reports, we identified the dark-coniferous forest, the mixed coniferous and broadleaf forest, and oak patches as suitable habitats (SH) for the monkeys. Summer grazing lands (SGL), which were made by local people cutting and burning the dark-coniferous forest at the high altitude belt, replaced SH. To have a general view of the status of the SH in Yunnan, we estimated the areas of SH and SGL from satellite images in 1997, and compared with areas estimated from aerial photo-based maps (ca. 1958). The work resulted in: 1) the area of SH was 4,169 km(2) in 1997; 2) SGL was 1,923 km(2); 3) during the past 40 years, the area of SH decreased by 31% (1,887 km(2)), and SGL increased by 204% (1,291 km(2)); and 4) the mean size of forest patches decreased from 15.6 to 5.4 km(2). In addition, the area of SGL is positively correlated to local human population (R-2 greater than or equal to0.53), implying that the reduction and fragmentation of habitat for Rhinopithecus bieti is a result of population growth of humans, who mostly employ traditional modes of production. Only 11 monkey groups remained in the changing habitat. Considering that forests at lower elevation were also encroached upon by farmlands in a similar way, the forest ecosystem is highly threatened. The destruction will continue unless there is a change in the mode of production in the region.