慢病毒载体导入种蛋内鸡胚技术研究

种蛋内鸡胚含有潜在的胚胎干细胞(BCs)或原生殖细胞(PGCs),是目前主要的转基因鸡研究方法。采用绿色荧光蛋白(GFP)基因的pLenti6/v5-DEST-GFP慢病毒表达载体,白来航鸡与伊萨鸡种蛋,结合种蛋赤道面开窗专利技术,对含这两种细胞胚的转基因技术进行了比较研究:转染白来航鸡囊胚,孵化13天时,GFP基因的PCR检出率为64.7%,孵化率极低;转染孵化72h伊萨鸡胚血液循环中PGCs,实验蛋孵化率为35.0%,在出壳后死亡的3只小鸡肝脏中,GFP基因的PCR检出率为100%,存活的4只鸡中有3只在12月龄的血液样品中,经PCR扩增出了GFP基因;转染孵化72～79h白来航鸡胚PGCs,7批次实验的平均孵化率为21.1%,能在赤道面窗口注射胚的种蛋比率,以73～77h胚龄的最高,为75.0%～92.9%,注射病毒组出壳雏鸡血液DNA中,GFP基因PCR检出率为44.4%。两种方法比较,PGCs方法在实验蛋孵化率、胚定位在赤道面窗口率等方面有较强优势。因此为种蛋内胚细胞的转基因鸡技术研究提供了系统、可操作性强的方法。     

侧脑室注射微量吗啡和纳络酮对小鼠记忆保持的影响

实验观察侧脑室注射微量吗啡和纳络酮对小鼠记忆保持的影响.结果表明:侧脑室注射吗啡后,小鼠从迷宫始点走到终点所需的时间显著延长,出现错误的次数增多,且具有一定的量效反应关系。这一作用可被纳络酮所阻断。而纳络酮对记忆的增强作用又可被吗啡所抵消。本实验提示中枢神经系统里的内源性阿片肽很可能参与记忆巩固的调制。     

鸡发育过程肌组织核提抽物结合活性与AChRγ亚基基因持续表达的关系

烟碱样乙酰胆碱受体 ( n ACh R)是由 4种亚基组成的五聚体 .哺乳类动物出生后γ亚基由ε亚基取代 ,迄今为止鸡 n ACh Rγ基因是否存在上述置换规律尚无定论 .为探索发育过程鸡骨骼肌n ACh R基因表达是否存在γ/ε亚基的置换及其机制 ,采用 RT- PCR技术和凝胶阻滞试验检测了鸡胚发育 9d至出生后 6周小鸡 γ基因表达的动力学及骨骼肌核抽提物的 DNA结合活性 .RT-PCR检测结果显示 ,在鸡胚发育 9d至出生后 6周的雏鸡骨骼肌组织均检出有 γ亚基 m RNA转录 .提示与哺乳类不同 ,出生前后鸡骨骼肌组织 ACh Rγ亚基基因持续表达 ,不存在 γ/ε亚基的置换表达规律 .以 γ基因 - 2 60 /- 2 4 0 (含 E盒与 M- CAT盒重叠序列 )和 - 2 39/- 50 (含 M- CAT盒及 GC富含区 )片段为探针 ,分别与鸡胚发育 9d至出生后 2周小鸡骨骼肌的核抽提物进行凝胶阻滞试验 .在发育各阶段的骨骼肌核抽提物中均有识别 - 2 39/- 50片段的结合活性存在 ,但在出生后 2周小鸡骨骼肌的核抽提物中未检出 - 2 60 /- 2 4 0结合活性 .结果提示 ,在出生后第 1 4d的肌核抽提物中存在的、识别并结合 - 2 39/- 50片段的活性物质与鸡 ACh Rγ基因在出生后持续表达有关 .     

鸡种系嵌和体的研制及其AFLP检测

利用密度梯度离心等方法从孵化51-56 h的石歧杂鸡胚血液中提取PGCs,用自制的玻璃注射针将PGCs注射到孵化2.5 d的H系受体鸡胚中制备种系嵌和体鸡;通过筛选AFLP引物建立起家禽嵌和体的AFLP检测方法;经检测20个发育的PGCs受体鸡胚中有８个种系嵌和体,嵌和率为40%。     

吗啡对鸡胚胎发育的影响

目的：研究吗啡对胎动、心率、孵化率、孵化时间、雏鸡体重等的影响。方法：以气室给药的方式给鸡胚注射吗啡,记录胎动、心率、孵化率、孵化时间、雏鸡体重。结果：吗啡可以缩短雏鸡的孵化时间,降低雏鸡的孵化率,并导致雏鸡出现运动障碍;20mg／kg吗啡剂量和12—16胚龄的给药时间,鸡胚孵化率最高,残疾率最低;吗啡导致胚胎心率加快,胎动减少（P〈0．05）。结论：吗啡对胚胎发育有损伤作用,损伤程度与吗啡剂量和给药时间有关。     

阻断NMDA受体可增强皮质酮对海马脑源性神经营养因子表达的抑制:cAMP反应元件结合蛋白的作用

高浓度的皮质酮可引起海马形态与功能的损伤,其中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) 表达的改变在海马形态与功能损伤中扮演重要角色。本实验的目的是观察单次皮下注射皮质酮后海马内BDNF-mRNA、前 体蛋白及成熟型蛋白表达的改变,并观察N-甲基-D-天冬氨酸(N-methyl-D-aspartate NMDA)受体阻滞剂MK801对皮质酮 作用的影响。实验结果显示,单次皮下注射皮质酮2 mg／kg,3 h后海马内BDNF mRNA、前体蛋白及成熟型蛋白的表达 均降低;MK801(0．1 mg／kg)对皮质酮的这一作用有增强效果。单独给予皮质酮或注射MK801 30 min后再给予皮质酮, 均能明显降低海马中cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)的磷酸化水平,MK801与 皮质酮联用时CREB的磷酸化水平降低更为显著(与单独给予皮质酮相比,P<0．05)。实验结果提示,CREB磷酸化水平降 低可能是皮质酮引起海马BDNF表达减少的重要中间环节,阻断NMDA受体可加强皮质酮降低BDNF表达的效应。     

鸡胚脑细胞谷氨酸释放的模拟微重力效应

用回转器旋转鸡胚蛋研究对脑细胞的模拟微重力生物效应.采用连续荧光法测量孵化10 d（E10）和孵化13 d（E13）鸡胚的脑细胞谷氨酸的初始释放速率、在KCl去极化及单个电脉冲刺激后的释放速率和释放量以及谷氨酸的含量,并对旋转处理组和静止对照组进行了比较.结果如下：旋转组和对照组脑细胞的谷氨酸初始释放速率没有显著差异,E10鸡胚经24 h旋转后,在KCl刺激下脑细胞的谷氨酸释放速率和释放量皆高于对照组,经4 h旋转后谷氨酸含量显著增高（P＜0.01）;但旋转24 h的E13鸡胚上述指标皆无显著改变,表明微重力对鸡胚脑细胞神经递质释放的影响与胚龄有关.鸡胚脑细胞在电脉冲刺激下谷氨酸释放的动态过程表明：脉冲电场引起的谷氨酸释放与细胞内钙离子迅速增加有关.     

胚胎发育早期接种人血清白蛋白诱导鸡的免疫耐受

外源基因的引入及表达常会引发宿主动物强烈的免疫应答,导致蛋白产量的下降或引起炎症反应。本研究试图通过在胚胎发育早期接种异源抗原诱导动物产生免疫耐受来解决这些问题。为了诱导鸡产生免疫耐受,将人血清白蛋白（Human serum albumin,HSA）通过胚胎血管微注射的方式接种到发育65—67h的鸡胚血管中,接种剂量为50μg;通过卵黄注射的方式接种到发育3—7d的鸡胚卵黄中,接种剂量为100μg。孵出的小鸡在3周龄时按照常规免疫方法再次接种同种抗原,收集不同时期的血清样本,用酶联免疫吸附试验（Enzyme-linked immunosorbent assay,ELISA）检测血清中抗-HSA抗体水平。结果表明,两种接种方式均能诱导小鸡产生免疫耐受：在65-67h的胚胎中通过血管微注射法接种抗原诱导小鸡产生免疫耐受的比率为64．52％;通过卵黄注射接种抗原诱导小鸡产生免疫耐受的最佳接种时间为发育的第6d,第5、7d接种对后期血清中抗-HSA抗体形成也有一定抑制作用,但是维持耐受的时间较短,第3、4d接种抗原对诱导小鸡免疫耐受的效果不明显[动物学报51（5）：845—851,2005]。     

多氯联苯对鸡胚原始生殖细胞的损伤作用

目的：本实验研究了PCB以及雌二醇（E2）,睾酮（T）和雌激素受体阻断剂克罗米酚（clomiphen)对5日龄鸡胚性朱中原始生殖细胞（PGC）形态和数量的影响,并对胚胎性腺受损伤程度进行了评价。方法：在入孵前将PCB（Aroclor 1254)油剂注入海兰种蛋卵黄内,实验组中PCB的剂量分别为1,10,100ug/枚;E2和T油剂注射剂量均为10,100ug/枚;克罗米酚,克罗米酚和Aroclor 1254均为100ug/枚,体积均为100ul;对照组注射等量花生油,孵化温度为38度,相对湿度60％,孵化111－120h后取出胚胎进行全固定,经石蜡切片和高碘酸席夫试剂染色后观察性腺中的PGC。结果（1）与对照组相比,PCB延缓鸡胚发育,但是鸡胚死亡率与PCB的剂量不呈现剂量依赖关系,最大死亡率在PCB 1ug/枚组;（2）性腺中的PGC的数量随PCB注射剂量的升高而显著降低,而且左侧性腺比右侧性腺明显,在PCB 100ug/枚组的性腺中,PGC发生了空泡化和核固缩甚至成为空洞;（3）PCB可以导致鸡胚性腺中PGC的损伤程度显著加强;（4）E2,T和克罗米酚处理后,对性腺中的PGC没有显著影响;（5）注射PCB和克罗米酚后,对性腺中PGC的影响与Aroclor 100ug/枚组的结果相同,结论：PCB对鸡胚生殖的影响早在PGC定居性腺就开始了,且对PGC的影响只表现其毒性作用,并没有通过类性激素作用影响PGC的形态和数量。     

回转器旋转对鸡胚脑细胞内游离Ca~(2+)水平的影响

采用ｆｕｒａ－２双波长荧光法,测定了孵化第６至１６天鸡胚脑细胞内游离Ｃａ２＋的浓度。并且利用回转器模拟微重力的生物效应,研究了不同胚龄鸡胚旋转不同时间后,脑细胞内游离Ｃａ２＋水平的变化。结果表明,孵化第８至１６天鸡胚旋转不同时间后,其脑细胞内游离Ｃａ２＋浓度均有所下降,其中第１０天鸡胚旋转４或７小时及第１３天鸡胚旋转２４小时后,脑细胞内Ｃａ２＋浓度比对照组显著降低（ｐ＜０．０１）。第１０天和１３天鸡胚分别旋转４和２４小时后再静置恢复相同时间,细胞内Ｃａ２＋浓度均有所增加,但仍低于对照组水平。结果显示,回转器旋转引起的鸡胚脑细胞内游离Ｃａ２＋水平降低是可逆的     

Plasma corticosterone in American kestrel siblings: effects of age,hatching order,and hatching asynchrony

Although it is well documented that hatching asynchrony in birds can lead to competitive and developmental hierarchies, potentially greatly affecting growth and survival of nestlings, hatching asynchrony may also precipitate modulations in neuroendocrine development or function. Here we examine sibling variation in adrenocortical function in postnatally developing, asynchronously hatching American kestrels (Falco sparverius) by measurements of baseline and stress-induced levels of corticosterone at ages 10, 16, 22, and 28 days posthatching. There was a significant effect of hatching order on both baseline and stress-induced corticosterone levels during development and these effects grew stronger through development. First-hatched chicks exhibited higher baseline levels than later-hatched chicks throughout development and higher stress-induced levels during the latter half of development. Furthermore, there was significant hatching span (difference in days between first- and last-hatched chicks) x hatching order interaction on both baseline and stress-induced corticosterone levels during development. Hatching span was also positively correlated with both measures of corticosterone and body mass in first-hatched chicks, but was negatively correlated with these factors through most of the development in last-hatched chicks. It is known that hatching asynchrony creates mass and size hierarchies within kestrel broods and we suggest that hierarchies in adrenocortical function among siblings may be one physiological mechanism by which these competitive hierarchies are maintained.     

Effects of prenatal exposure to a 50‐Hz magnetic field on one‐trial passive avoidance learning in 1‐day‐old chicks

We investigated memory impairment in newly hatched chicks following in ovo exposure to a 50‐Hz magnetic field (MF) of 2 mT (60 min/day) on embryonic days 12–18. Isolated and paired chicks were used to test the effect of stress during training, and memory retention was tested at 10, 30, and 120 min, following exposure to a bitter‐tasting bead (100% methylanthranilate). Results showed that memory was intact at 10 min in both isolated and paired chicks with or without MF exposure. However, while isolated chicks had good memory retention levels at 30 and 120 min, those exposed to MF did not. The results suggest a potential disruption of memory formation following in ovo exposure to MF, with this effect only evident in the more stressed, isolated chicks. Bioelectromagnetics 31:150–155, 2010. © 2009 Wiley‐Liss, Inc.     

Body condition,hormonal correlates and consequences for survival in common tern chicks

Although sibling competition in avian species has been extensively studied, the proximate mechanisms remain largely unknown. Recent research proposed that steroid hormones, in particular testosterone (T) and corticosterone (CORT), might play a role either in promoting competitive behavioral displays or in response to chronic nutritional stress accompanied by a sustained competitive situation. Here, we examine body condition, endogenous T levels and fecal glucocorticoid metabolites (FGM) as non-invasive measures of CORT in sibling broods of wild common tern chicks (Sterna hirundo) during three post-natal developmental stages. In this species, distinct within-brood size asymmetries are imposed by an asynchronous hatching interval, and sexes show slightly different growth patterns. First-hatched (a-)chicks were in better condition than their later-hatched sibling (b-chick). FGM levels inversely covaried with condition and were elevated at the end of pre-fledging development. T levels of a- and b-chicks changed with age, although the direction of the changes differed, with b-chicks eventually having higher levels than their older siblings. Survival to fledging was not associated with FGM but with T levels, which tended to be higher in surviving chicks. Our results are discussed with regard to how plasticity in steroid hormones could be involved in mediating sibling competition in common terns.     

Rapid inhibitory effect of corticosterone on histamine release from rat peritoneal mast cells.

Glucocorticoids are steroids endowed with powerful anti-inflammatory properties, which are routinely believed to require several hours to take effect through modulation of gene expression. Our recent report has shown that glucocorticoids could inhibit allergic reaction within 10 minutes, which the classical genomic mechanism could not explain. Histamine is thought to be one of major mediators in the allergic reaction, and IgE-mediated histamine release from mast cells plays a pivotal role in allergic diseases. Here, we have determined a rapid effect of corticosterone on histamine release from rat peritoneal mast cells, using fluorometric assay. The results showed that corticosterone could inhibit antigen-induced histamine release from rat peritoneal cells within 15 minutes (p<0.05), which could be mimicked by membrane-impermeable BSA conjugated corticosterone (p<0.05). Neither glucocorticoid nuclear receptor antagonist nor protein synthesis inhibitor could block the rapid action (p<0.05). The study provided evidence that nongenomic mechanism might be involved in rapid effect of glucocorticoids on mast cells in allergic disease.     

Variation in faecal corticosterone metabolites in an Arctic seabird,the Little Auk (Alle alle) during the nesting period

Glucocorticoids participate in the control of whole body homoeostasis and an organism’s response to stress. Corticosterone, which is the principal glucocorticoid in birds, has been shown to increase in response to different energetic demands and perturbations that individuals have to cope with. In this study, a non-invasive method to examine the corticosterone secretion by measuring faecal corticosterone metabolites (FCM) has been established for an Arctic seabird, the Little Auk (Alle alle). A group-specific immunoassay was successfully validated for adults and chicks using an adrenocorticotropic challenge test. Then, FCM levels were investigated under different energetic and physiological demands, determined by weather conditions, week of chick rearing in adults, and age in chicks. The amount of rainfall had no effect on FCM levels in adults, whereas it negatively affected FCM levels in chicks. There was no variation in FCM concentrations among weeks of chick rearing in adults. In chicks, the FCM levels increased with age. Moreover, chicks with higher FCM levels had lower body mass and fledged later than chicks with lower FCM levels. This study demonstrates that environmental stress such as poor weather conditions can trigger significant changes in corticosterone levels in seabird chicks. Furthermore, the results indicate that corticosterone may be involved in the physiological and behavioural adjustments necessary for successful fledging and post-fledging survival.     

Pre- and post-hatching effects of corticosterone treatment on behavior of the domestic chick

We investigated the effect of 60 ìg of corticosterone administered to domestic chicks either before or after hatching on the behavioral response to isolation in a novel arena and performance in a task involving the simultaneous identification of food and detection of a predator (overhead silhouette of a hawk moving overhead). Following release into a novel arena, chicks treated with corticosterone at 18 days of incubation emitted more distress vocalizations. In contrast, no difference in the number of vocalizations was found between chicks treated with corticosterone at day 1 post-hatching and controls. Behavior in the home cages was generally similar across treatments, though chicks treated with corticosterone at 18 days of incubation slept more than control chicks. While searching for grain against a background of pebbles, chicks treated with corticosterone at embryonic day 18, but not chicks treated on day 1 post-hatching, took longer to detect the overhead image of a predator than did controls. Corticosterone treatment at both ages increased the rate of pecking at grains and pebbles. Our findings support work on other birds indicating that corticosterone treatment during incubation influences stress reactivity. The impairment in predator detection in chicks treated with corticosterone on day 18 of incubation appears to be caused by the known effects of corticosterone treatment at this age in preventing the development of lateralization of the thalamofugal visual projections. This further supports the hypothesis that brain lateralization provides an advantage in performing more than one task simultaneously.     

Morphine-induced increase in D-1 receptor regulated signal transduction in rat striatal neurons and its facilitation by glucocorticoid receptor activation: Possible role in behavioral sensitization

One month (but not 1–3 days) after intermittent morphine administration, the hyperresponsiveness of rats toward the locomotor effects of morphine and amphetamine was associated with an increase in dopamine (DA) D-1 receptor-stimulated adenylyl cyclase activity and enhanced steady state levels of preprodynorphin gene expression in slices of the caudate/putamen and nucleus accumbens. Such an enduring increase in postsynaptic D-1 receptor efficacy also occurred in cultured γ-aminobutyric acid (GABA) neurons of the striatum obtained from rats prenatally treated with morphine. Interestingly, in vitro glucocorticoid receptor activation in these cultured striatal neurons by corticosterone potentiated this neuroadaptive effect of prior in vivo morphine exposure. Since activation of glucocorticoid receptors by corticosterone did not affect D-1 receptor functioning in cultured neurons of saline-pretreated rats, prior intermittent exposure to morphine (somehow) appears to induce a long-lasting state of corticosterone hyperresponsiveness in striatal neurons. Therefore, DA-sensitive striatal GABA neurons may represent common neuronal substrates acted upon by morphine and corticosterone. We hypothesize that the delayed occurrence of these long-lasting morphine-induced neuroadaptive effects in GABA/dynorphin neurons of the striatum is involved in the enduring nature of behavioral sensitization to drugs of abuse and cross-sensitization to stressors. Special issue dedicated to Dr. Eric J. Simon.     

Prior exposure to a single stress session facilitates subsequent contextual fear conditioning in rats. Evidence for a role of corticosterone

Previous studies showed that exposure of rats to chronic restraint stress for 21 days enhances subsequent contextual fear conditioning. Since recent evidence suggest that this effect is not dependent on stress-induced neurodegenerative processes, but to elevated training-elicited glucocorticoid release in chronically stressed animals, we aimed to explore here whether a single exposure to restraint stress, which is not expected to induce neuronal damage, would also affect contextual fear conditioning. We also questioned whether post-training corticosterone levels might be associated with any potential effect of stress on fear conditioning. Adult male Wistar rats were exposed to acute restraint stress for 2 h and, two days later, trained in the contextual fear conditioning task, under training conditions involving either moderate (0.4 mA shock) or high (1 mA shock) stress levels. The results showed that acute stress enhanced conditioned freezing at both training conditions, although data from the 1 mA shock intensity experiment only approached significance. Stressed animals were shown to display higher post-training corticosterone levels. Furthermore, the facilitating effect of prior stress was not evident when animals were trained in the hippocampal-independent auditory-cued conditioning task. Therefore, these findings support the idea that stress experiences preceding exposure to new types of stressors facilitate the development of contextual fear conditioning. They also indicate that not only repeated, but also a single exposure to aversive stimulation is sufficient to facilitate context-dependent fear conditioning, and suggest that increased glucocorticoid release at training might be implicated in the mechanisms mediating the memory facilitating effects induced by prior stress experiences.     

Corticosterone decreases the efficacy of adrenaline to affect passive avoidance retention of adrenalectomized rats

Short-term (48h) adrenalectomy (ADX) resulted in a deficit in the retention of a passive avoidance response. An inverted U-shaped dose-response relationship was found following immediate post-learning administration of adrenaline (A). A in a dose range of 0.005 - 5 micrograms/kg s.c. facilitated later retention. While corticosterone (CS) replacement alone had no effect, pretreatment with CS (300 micrograms/kg) was followed by a shift in the dose-response curve of A in ADX rats. Ten thousand times higher doses of A were required to improve retention behavior. Administration of the potent synthetic glucocorticoid dexamethasone failed to affect the responsiveness to A. It is concluded that corticosterone decreases the efficacy by which adrenaline affects later retention behavior of ADX rats. The specificity of corticosterone in this interaction suggests the involvement of the corticosterone receptor system which has its predominant localization in hippocampal neurons.