【作者】 钱静；

【导师】 王玉玮；

【作者基本信息】 山东大学 ， 儿科学， 2008， 博士

【摘要】 背景孕母与胎儿的关系十分密切,任何影响母亲健康的因素都有可能同时影响胎儿发育。近年来社会心理因素在妊娠过程中的作用逐渐被人们所重视。国外学者通过多种方法,制造出多种能够产生负性情绪的动物模型达到类似社会心理应激的效果,发现孕期应激可以使其仔鼠恐惧行为加剧,并有着与人类焦虑症患者相似的行为、生化和解剖学的特点,因此,目前出生前有应激史的仔鼠已经成为研究焦虑症的动物模型。探讨其发病机制以及如何逆转其焦虑的发生将为人类焦虑症的预防和治疗提供重要的理论和实践依据。另外,关于良好的孕期环境是否可减少其仔鼠的恐惧行为,目前尚无研究报道。杏仁核是恐惧回路的重要组成部分,条件恐惧的细胞分子生物学机制主要集中于杏仁核中兴奋性谷氨酸通路和抑制性γ-氨基丁酸(GABA)通路的相互作用,涉及细胞电生理的改变、基因的表达和蛋白质的合成。尽管GABA能中间神经元只占了神经元中的一小部分,但这些神经元在兴奋性神经元间起着有力的抑制性的使兴奋性神经元同步化的作用,同时在受到刺激后起着有力的前馈和反馈的抑制性突触后电位的作用。胃泌素释放肽(gastrin-releasing peptide,GRP)及其受体(GRPR)被一些研究者认为与恐惧记忆高度相关。体外研究发现,GRPR基因敲除小鼠的杏仁核切片中同步性抑制水平大大降低,并且长时程增强效应(long term potentiation,LTP)增强,这些小鼠在Morris水迷宫中的空间记忆不会受到损害(海马依赖的空间记忆),但却表现出对巴甫洛夫经典条件恐惧中对听觉线索(杏仁核依赖)和情景线索(杏仁核海马均依赖)的长期恐惧记忆的加强和延长,同时表现出与恐惧记忆有关的神经回路皮层LTP效应加强,从而证实GRP对恐惧记忆起抑制性负反馈作用。然而,另有研究显示,无论给予海马CA1区或基底外侧杏仁核注射GRPR拮抗剂均可损害厌恶性条件形成,提示GRP对恐惧记忆起促进性作用。因此,GRP及其受体与条件恐惧记忆的关系尚无肯定结果。“丰富环境”(environmental enrichment,EE)被定义为复杂的、无生命物与社会刺激的复合体。环境对大脑功能及行为的影响已得到广泛重视。有研究显示丰富环境不仅可以明显提高动物的记忆和学习能力,还能减少其焦虑行为。但是目前尚无研究显示丰富环境是否影响大脑GRPR的表达。目的1.运用已被广泛用于评价大鼠恐惧水平的防御退缩实验、旷场实验,研究孕期不同环境对不同日龄仔鼠恐惧行为的影响。2.采用经典的孕期应激模型,研究仔鼠的恐惧行为与杏仁核、海马GRPR阳性细胞数量、GRPR信息RNA及蛋白表达水平的关系,试图进一步说明条件恐惧过程中抑制性神经通路所起的作用。3.研究丰富环境是否影响仔鼠杏仁核、海马GRPR阳性细胞数量、GRPR信息RNA及蛋白表达,进一步阐明丰富环境作用的分子机制。研究方法1.孕期不同环境对不同日龄仔鼠恐惧行为的影响:将初次妊娠11天的孕鼠随机分为:应激组、丰富环境组和对照组,每组各4只,1只/箱。应激组:将妊娠11—21天的孕鼠置于透明塑料的固定器(直径7cm,长19cm)中并给予强光照射45分钟/次,3次/天(分别在10:00,13:00和16:00点)。丰富环境组:将孕鼠置于长×宽×高为80×60×40 cm的特制饲养箱中,内设:转笼、掩体、各种颜色的塑料玩具以及其它小装置如秋千、隧道等;其中转笼和掩体是固定的,其他的玩具和小装置则每星期更换。对照组不给予刺激。出生当天将每窝仔鼠调整到10只,生后21天断奶,4只/箱,标准环境喂养,每组16只雄性仔鼠分别于生后25天、45天和60天进行旷场实验和防御退缩实验测试。2.丰富环境对孕期应激仔鼠恐惧行为的影响及其机制的研究:断奶后,将孕期应激雄性仔鼠40只随机分为2组,每组20只:应激丰富环境组(SE),置于特制饲养箱中,10只/箱;应激对照组(SC),标准环境喂养,4只/箱。另有对照组雄性仔鼠40只分组方法同上,分别称为标准丰富环境组(CE)和标准对照组(CC)。生后60天进行防御退缩实验测试。测试结束后,每组6只过量麻醉后灌注固定取脑,分别进行尼氏染色和GRPR单标免疫荧光染色,计数海马各区、杏仁核各核团以及相应大脑皮层GRPR阳性细胞数;其余仔鼠直接断头取脑,在冰上快速分离双侧海马、杏仁核及相应体积的大脑皮层,应用实时定量RT-PCR法(Real-time Quantitative RT-PCR)检测各部位GRPR信息RNA的表达;应用免疫印记(Western blot)检测各部位GRPR蛋白的表达。结果1.孕期不同环境对不同日龄仔鼠恐惧行为的影响在旷场实验中,各日龄孕期应激仔鼠均较相应对照组中央格停留时间延长,总穿格数及站立次数减少(all p＜0.05),提示孕期应激可以减少其仔鼠对陌生环境的探究行为。与之相反,生后25天时丰富环境组仔鼠较对照组总穿格数增加(p＜0.05),而中央格停留时间缩短(p＜0.05),至生后45天、60天上述差别消失(all p＞0.05),说明孕期给予丰富的环境仅可以增加其幼鼠的探究行为。在防御退缩试验中,生后25天时,孕期应激仔鼠较对照组潜伏期延长(p＜0.05);生后45天时,不仅潜伏期延长(p＜0.05),自小室退出次数亦减少(p＜0.05);而生后60天时,在上述表现的基础上,给予束缚应激刺激后其在小室内的时间较相应对照组明显延长(p＜0.05);提示孕期应激刺激可以导致子代的恐惧行为增加,且随着年龄增长逐渐加剧,给予束缚应激刺激可加剧其恐惧。与之相反,生后25天时,丰富环境组仔鼠较对照组潜伏期缩短,自小室退出次数增加,说明孕期丰富环境可以减少幼年仔鼠的恐惧行为并增加其探究行为。2.丰富环境对孕期应激仔鼠恐惧行为的影响经给予丰富环境刺激,在生后60天进行的防御退缩试验中,应激丰富环境组(SE)较应激对照组(SC)潜伏期缩短(p＜0.05),出小室的次数增多(p＜0.05),给予束缚应激不再增加其在小室内的时间(p＜0.05),提示丰富环境可以纠正孕期应激仔鼠异常的恐惧行为,并提高其对应激刺激的适应能力。3.出生前后不同环境对杏仁核、海马及大脑皮层GRPR阳性细胞数量的影响孕期应激仔鼠(SC组)海马CA1、CA3、DG区,杏仁外侧核(LA)、中央核(CE)、基底外侧核(BLA)以及相应大脑皮层GRPR阳性细胞计数较标准对照组无显著性差异(all p＞0.05);而应激丰富环境组(SE)及标准丰富环境组(CE)在海马各区及杏仁核各核团GRPR阳性细胞计数均较相应对照组明显增多(all p＜0.05)。4.出生前后不同环境对杏仁核、海马及大脑皮层GRPR信息RNA表达水平的影响孕期应激仔鼠(SC组)海马、杏仁核以及相应大脑皮层GRPR信息RNA表达水平较CC组无显著性差异(all p＞0.05);而SE及CE组海马和杏仁核GRPR信息RNA表达水平较SC和CC组明显提高(all p＜0.05)。5.出生前后不同环境对杏仁核、海马及大脑皮层GRPR蛋白表达水平的影响孕期应激仔鼠(SC组)海马、杏仁核以及相应大脑皮层GRPR蛋白表达水平较CC组无显著性差异(all p＞0.05);而SE及CE组海马和杏仁核GRPR蛋白表达水平较SC和CC组明显提高(all p＜0.05)。结论1.本研究再次证实,孕期给予制动加强光照射应激刺激可以导致子代的恐惧行为增加,且随着年龄增长逐渐加剧,给予束缚刺激可以加剧其恐惧,这与人类焦虑症常在幼年发病,可持续至青春期及成年,表现为过多的恐惧和对不良刺激的回避基本吻合,说明该动物模型作为焦虑症的实验模型基本合乎要求。2.本次研究首次发现,孕期给予丰富的环境可以降低其幼鼠的恐惧水平,增加其探究行为,但是这一优势于生后45天即消失,可能与大鼠的恐惧水平随年龄增长而增加有关,具体机制有待进一步探讨。3.本次研究首次发现,孕期应激并不影响仔鼠海马、杏仁核以及相应大脑皮层GRPR阳性细胞数量、GRPR信息RNA和蛋白表达水平,其机制有待进一步研究。4.本次研究首次发现,丰富环境可以增加海马CA1、CA3、DG区,杏仁核LA、CE、BLA的GRPR阳性细胞数,并提高海马、杏仁核GRPR信息RNA及蛋白表达,同时伴有恐惧水平的降低,支持GRPR作为一种负反馈来调节恐惧记忆。提示丰富环境可能通过提高杏仁核和海马GRPR表达水平来改善孕期应激仔鼠过度的恐惧行为,从而可能成为一个有效而低风险的早期干预手段。5.本次研究结果还提示,先天性GRP及其受体缺陷的人可能具有焦虑症的易患素质,可能是部分病人易患焦虑症的原因。更多还原

【Abstract】 BackgroundIt is well known that the relationship between mother and fetus is significant, and many factors during pregnancy can lead to physical malformations or behavioral dysfunctions. In the past few years, the effects of social-psychological factors during pregnancy have gradually aroused the attention of the public. In order to study the effect of prenatal stress on their offspring, many kinds of animal models have been made, and many studies have shown that prenatal stressed (PS) offspring exhibited more fearful behavior in behavioral tests. Furthermore, as showing behavioral, biochemical and anatomical similarities to anxious humans, the PS rat has become an animal model to study pathological anxiety. It is obviously that exploring the pathogenesis and prevention of abnormal fearful behavior of PS rat maybe contribute to the prevention and treatment of human anxiety disorders. Besides, the effects of environmental enrichment during pregnancy on fearful behavior of their offspring have been less documented and remain questionable.The amygdala is a key component of the neural circuitry of fear—both innate and learned in humans and in simpler vertebrate experimental animals. Cellular and molecular mechanisms of conditioned fear are focused on the interaction between positive glutamine pathway and inhibitory GABAergic pathway, involving in the electrophysiological changes, gene expression and synthesis of protein. Although GABA interneurons account for a small portion of neurons, they play a crucial role in inhibiting the excitatory neurons and regulating the complex interactions among principal cells, moreover, inhibitory postsynaptic potentials elicited from GABA interneurons has a robust effect of feed-forward and feedback.GRP and GRP receptor (GRPR) are distributed throughout the mammalian central nervous system. Gastrin-releasing peptide receptors (GRPR) have an important role in regulating amygdala-dependent, fear-related learning. GRPR-deficient mice showed decreased inhibition of principal neurons by interneurons in slices, enhanced long-term potential (LTP); these mice performed normally in hippocampus-dependent Morris maze, but showed greater and more persistent long-term fear memory in Pavlovian auditory cue fear conditioning, an amygdala-dependent task, and contextual fear conditioning which depend both on the amygdala and the hippocampus, which provide genetic evidence that GRP and its neural circuitry operate as a negative feedback regulating fear memory. However, both intrahippocampal infusion and microinjected into the basolateral amygdala of the bombesin/gastrin-releasing peptide antagonist RC-3095 impair formation of aversive memory, indicating that GRP system(s) can significantly enhance fear memory. In conclusion, the relationship between GRP, GRPR and conditioned fear memory remain questionable.Environmental enrichment (EE) is defined as a combination of "complex inanimate objects and social stimulation". The importance of the environment in brain regulation, behavior and physiology has long been recognized in the biological, social and medical sciences. Animals maintained under enriched conditions clearly have better memory, learning abilities and less anxiety-like behavior than those housed under standard conditions. However, the effects of environmental enrichment on the expression of GRPR in the brain have been less documented.Objective1. To investigate the effects of different stimulation during pregnancy on fearful behavior of offspring at 25, 45 and 60 days of age in the open field test and defensive withdrawal test.2. To investigate the relationship between fearful behavior and the number of GRPR positive cells, levels of GRPR mRNA and protein in the amygdala and hippocampus of prenatal stressed offspring.3. To investigate the effect of postnatal environmental enrichment on the number of GRPR positive cells, expression of GRPR mRNA and protein in the amygdala and hippocampus of PS offspring, and to explore the molecular mechanisms of environmental enrichment.Method1. On gestational days 11, primigravid female rats were randomly assigned to three groups: enriched group, stressed group and the control group, n=4 per group. All animals were individually housed. Prenatal treatments were performed daily on gestational days 11-21. The enriched dams were kept in specially designed cages (80 cm long×60 cm wide×40 cm high) that were equipped with a running wheel, a shelter, plastic color toys and small constructions such as chain and swing, tunnels. Throughout the enrichment periods, the shelter and running wheel were kept in the cage, while the toys and constructions were changed once a week. The stressed dams were put into a narrow animal holder and exposed to bright light for 45 min three times a day (starting from 1000, 1300 and 1600 h). The control dams were left undisturbed. At birth, the litters were randomly culled to 10 pups within 24 h post delivery. The offspring were left undisturbed together with their mother in standard cages. Male and female offspring were weaned at postnatal day 21(P21), and only male offspring (n=16 per group) were used in the present study. Open field test and the defensive withdrawal test were performed at P25, P45 and P60.2. From P21, 20 pups of control or stressed dams (CC group, SC group) were kept in standard Plexiglas cages (60 cm long×40cm wide×25cm high, n = 4 per cage) and left undisturbed. The same number pups from control or stressed dams (CE group, SE group) were kept in specially designed cages (n = 10 per cage). Throughout the enrichment periods (P21-P60), the shelter and running wheel were kept in the cage, while the toys and constructions were changed once a week. Also once a week, the feeding boxes and water bottles were moved to different cage points to encourage foraging and explorative behaviors. On P60, all pups were tested in the defensive withdrawal and then sacrificed. Six rats per group used in the study of GRPR staining and Nissl staining received an overdose of chloral hydrate (400mg/kg, i.p.) and perfused with different solution. The other part was sacrificed by decapitation and the cortical portion, amygdala and hippocampus were dissected on ice. These brains were used in the study of Western blot and quantitative real-time detection PCR.Results1. Effects of different stimulation during pregnancy on fearful behavior of their offspring at 25,45 and 60 days of age.Open-field testPS offspring at any age had greater central grille time, decreased total cross number and stand number than their corresponding controls (all p<0.05) , showing that prenatal stress inhibited exploratory behavior of their offspring in novel circumstance. On the contrary, the prenatal enriched offspring at 25 days of age had increased total cross number (p<0.05) and shorter central grille time (p<0.05) than their controls, however, the difference between enriched and control groups disappeared at postnatal day 45 and 60, indicating enriched environment during pregnancy can only enhanced exploratory behavior of their infancy offspring.Defensive-withdrawal testWhen compare to the controls, PS offspring had greater latencies at P25, greater latencies and decreased number of exits from the chamber at P45, and greater latencies, decreased number of exits from the chamber and increased time in the chamber with restraint at P60 (all p <0.05), which indicating that PS offspring had more fearful behavior than their corresponding controls, and the fearful behavior was enhanced with increasing of age. On the contrary, the prenatal enriched offspring at 25 days of age had shorter latencies and increased number of exits from the chamber than their controls(all p <0.05), but the difference between enriched and control groups disappeared at postnatal day 45 and 60, indicating enriched environment during pregnancy decreased fearfulness and enhanced exploratory behavior of their infancy offspring.2. Effects of enriched environment treatment on fearful behavior of PS offspringPS offspring showed less fearfulness after maintained under enriched conditions, which was measured as a shorter latency and increased number of exits from the chamber without restraint, and was less affected by restraint stress(all p <0.05), which indicate that enriched environment treatment can reverse the abnormal behavior of PS offspring.3. Effects of pre and postnatal circumstance on the number of GRPR -positive cells in cortical portion, amygdala and hippocampusWhen compared to the control pups (CC group and SC group) respectively, pups maintained under enriched environment (CE group and SE group) exhibited significantly increase in density of GRPR -positive cells in amygdala nuclei and hippocampus areas (all p <0.001), but there were no significant difference between CE and SE groups or SC and CC groups (all p>0.05). No differences were found in density of GRPR -positive cells in the cortex containing M1, M2, RSA and RsGb areas among all groups [F(3,143)=0.34, p>0.05].4. Effects of pre and postnatal circumstance on the expression of GRPR mRNA in cortical portion, amygdala and hippocampusGRPR gene expression in the amygdala and hippocampus of CE group were up-regulated 136.47±48.75 and 146.30±37.46 when compared to CC groups (all p <0.001), and GRPR gene expression in the amygdala and hippocampus increased 129.25±39.51 and 151.74±43.38 fold respectively in SE group comparing with that in SC groups (1.22±0.39, 0.98±0.21) (allp<0.001). However, there were no significant difference between CE group and SE group (all p>0.05) or SC group and CC group (all p>0.05), besides, no differences were found in GRPR gene expression in the cortex containing Ml, M2, RSA and RsGb areas among all groups [F(3,39)=1.42, p>0.05].5. Effects of pre and postnatal circumstance on GRPR protein in cortical portion, amygdala and hippocampusGRPR protein expression in the amygdala and hippocampus of CE groups were up-regulated 1.06±0.12 and 1.29±0.23 when compared to CC groups(0.29±0.04, 0.29±0.04) (all p <0.001), and GRPR protein expression in the amygdala and hippocampus of SE groups (0.97±0.12,1.39±0.29) were increased when compared with that of SC groups (0.28±0.03, 0.28±0.05) (all p<0.001). However, there were no significant difference between CE group and SE group (all p>0.05) or SC group and CC group (all p>0.05), besides, no differences were found in GRPR protein expression in the cortex containing M1, M2, RSA and RsGb areas among all groups [F(3,31)=0.30, p>0.05].Conclusions1. Our studies provided more evidence that prenatal stress (put into a narrow animal holder and exposed to bright light) enhance fearfulness of their offspring, which can be significantly increased following acute restraint, and with increasing age, the fearful behavior was enhanced. These findings in PS rats are similar to the data from humans showing that the onset of pathological anxiety often manifests at a young age, persists throughout adolescence and continues into adulthood, exhibiting increased fearfulness, manifesting as avoidance of anxiety provoking situations. So this animal model can be used as a model for the study of anxiety disorders.2. We found for the first time that environmental enrichment during pregnancy only can decrease fearfulness and enhance exploratory behavior of their infancy offspring. Further research will be needed to elucidate the mechanism.3. We found for the first time that prenatal stress has no effect on the number of GRPR -positive cells, the expression of GRPR gene and protein in the cortical, amygdala and hippocampus of their offspring, the mechanism need to be explored.4. We found for the first time that enriched environment treatment can reverse the abnormal behavior of PS offspring, and increase the expression of GRPR in the amygdala and hippocampus, indicating GRP and its neural circuitry operate as a negative feedback regulating fear memory.5. In conclusion, the present studies provided evidence that GRPR and its neural circuitry operate of GABA interneurons as a negative feedback regulating fear memory. Postnatal environmental enrichment can reverse the enhanced fearfulness in adult PS offspring, which might be in part mediated by modulation of GRPR in the amygdala and hippocampus. Therefore, enriched environment treatment may become a new method to cure anxiety disorders. Besides, our study also suggested congenital defect of GRP and/or GRPR may lead to the onset of anxiety disorders.更多还原