【作者】 宣富君；

【导师】 张树义；

【作者基本信息】 华东师范大学 ， 生态学， 2012， 博士

【摘要】 蝙蝠具有众多独特的生物学特性。本论文围绕蝙蝠视觉、回声定位及其冬眠特性展开,分别就蝙蝠紫外视觉能力、语言基因Foxp2与回声定位相关核团及冬眠前后一类新兴过氧化物酶Peroxiredoxins进行了相关的脑区组织学比较研究：1：为揭示蝙蝠基于视锥细胞的紫外视觉能力,本研究对四种不同生态习性且具代表性的蝙蝠进行了双目紫外刺激实验。通过比较分析主视皮层Fos蛋白的表达差异,我们发现：在食虫类回声定位蝙蝠中,恒频大蹄蝠(Hipposideros armiger, CF)应该已经丧失了基于短波视蛋白的紫外视觉能力,1h刺激后在主视皮层,这个大脑图像处理中枢,相对于对照组并没有出现更多的Fos蛋白阳性信号,而调频小黄蝠(Scotophilus kuhlii, FM)却应该保留了这种紫外视觉能力,在其主视皮层出现了大量Fos蛋白的表达；相应地,在食果实的旧大陆蝙蝠中,洞栖棕果蝠(Rousettus leschenaultii)也丧失了这种紫外视觉能力,相对于对照组亦没有在主视皮层出现更多的Fos蛋白,而树栖犬蝠(Cynopterus sphinx)却应该也保留了这种紫外视觉能力,主视皮层出现大量信号。因此,本实验免疫组化数据在表明蝙蝠基于视锥细胞紫外视觉能力的同时,也进一步提供了翼手目可能的基于该紫外视觉的分布情况。而且,本研究结果与之前报道的分子进化推论相符合,即蝙蝠在进化过程中,可能由于感觉代偿及栖息环境差异,具有更高级别回声定位能力的恒频蝙蝠和洞栖果蝠它们的短视蛋白基因发生了基因缺失,丧失了紫外视觉能力；而调频蝙蝠和树栖果蝠却保留了,蝙蝠是另一类拥有基于视锥细胞紫外视觉能力的哺乳动物。2:Foxp2作为一种在发声及相关神经调控方面起重要作用的多功能转录因子,可能与蝙蝠回声定位能力的发展和进化有关。为此,我们运用原位杂交技术,对具有不同回声定位能力蝙蝠的两个代表性脑区——前侧扣带皮层(ACC)和嗅结节(Tu), Foxp2基因的差异性达表情况进行了进一步研究。其中,调查的5种四大类代表性蝙蝠物种包括食虫类具回声定位能力的马铁菊头蝠(恒频,CF)(Rhinolophus ferrumequinum),大蹄蝠(恒频,CF)(Hipposideros armiger),大足鼠耳蝠(调频,FM)(Myotis ricketti),旧大陆果蝠中的棕果蝠(Rousettus leschenaultii)(利用舌头敲击上颌发出超声)及犬蝠(Cynopterus sphinx)(不发超声)。结果表明：Foxp2在ACC脑区食虫类回声定位蝙蝠的表达量明显高于旧大陆果蝠,而在Tu脑区则后者较为丰富；另外,在各自分属的组内,物种间并没有出现明显差异。因此,本研究的结果不仅进一步支持了我们之前免疫组化的结果,也进一步表明：Foxp2在蝙蝠脑区的差异性表达除了可能与回声定位能力相关外,也很有可能是一个与旧大陆果蝠基于嗅觉系统食物选择能力相关的一个基因。更重要的是,本项工作可为后期进一步进行脑区功能实验,真正揭示Foxp2基因功能多样性提供可靠的形态学位点。3：哺乳动物的整个冬眠过程一般由多个延长的蛰伏期并伴随短时间的苏醒组成。期间,大脑,这个易受氧化应激损伤的部位,为抵御该过程因产能增温而产生的大量自由基ROS/RNS,机体自身必须得有一套功能强大的抗氧化系统。本研究运用免疫组织化学手段,第一次对冬眠动物大足鼠耳蝠Myotis ricketti冬眠前后40多个代表性脑区PrxⅢ, DJ-1及PrxI三个抗氧化物酶的分布及差异性表达情况进行了系统地比较研究。结果发现：PrxⅢ, DJ-1和PrxI在蝙蝠脑区的神经胶质细胞和神经元内均出现了不同层次的表达；且与活跃阶段相比,上述三蛋白酶整体表达量在冬眠阶段均有了显著的提高。这充分表明Peroxiredoxins这个过氧化物酶家族很有可能就是冬眠动物脑区最主要的抗氧化物。而且,通过比较冬眠动物与非冬眠模式动物脑区,主运动皮层、海马及黑质,三个分别与ALS, AD及PD发病机理密切相关核团Peroxiredoxins的表达差异分析,进一步揭示了这种冬眠动物特有抗氧化机制潜在的医用价值。最后,基于过氧化物酶Peroxiredoxins,我们对冬眠动物的抗氧化机制进行了探讨,推测这种特殊高效的抗氧化能力可能就是机体自身具有的一种脑组织预处理保护模式,而这种能力的获得可能还与蝙蝠长寿命的特性相关。更多还原

【Abstract】 Bats have a number of unique biological characteristics. This dissertation, on the topics of vision, echolocation and hibernation, details the comparative histological studies conducted on brain tissues of divergent bats to detect the cone-based UV vision, the relationship between language gene Foxp2and echolocation and an emerging class of peroxisome Peroxiredoxins in pre-and post-hibernation:1. We characterized Fos-like expression patterns in the primary visual cortex (VI) by binocular flicking stimulation with UV light to investigate cone-based UV vision in four bat species representing four lineages:Hipposideros armiger and Scotophilus kuhlii, insectivores using constant frequency (CF) or frequency modulation (FM) echolocation, respectively, and Rousettus leschenaultii and Cynopterus sphinx, cave-roosting and tree-roosting fruit bats, respectively. The optic centre processing the visual image, V1, appears more distinctly immunostaining in S. kuhlii and C. sphinx after1h of UV light stimuli while in H. armiger and R. leschenaultii, staining was no more distinct than in corresponding controls. Our immunohistochemical evidence supports differences in the distribution of cone-based UV vision in the order Chiroptera and supports our earlier postulate that due to possible sensory tradeoffs and roosting ecology, defects in the short wavelength opsin genes have resulted in loss of UV vision in CF but not in FM bats. In addition, fruit bats roosting in caves have lost UV vision but not those roosting in trees. Our results thus confirm that bats are a further mammalian taxon that has retained cone-based UV sensitivity in some species.2. Foxp2, a multi-functional transcription factor specially implicated in the development and neural control of vocalization, may have some relationship with the evolution and development of bats’echolocation. Here we employed in-situ hybridization to further investigate the divergent expressions of Foxp2in the anterior cingulate cortex (ACC) and olfactory tubercle (Tu), two representative brain areas of five species among four types of bats using or non-using different echolocation systems. Moreover, we compared these results from three insectivorous species with well-developed laryngeal echolocation (Rhinolophus ferrumequinum and Hipposideros armiger, bats emitting a long constant-frequency pulse, and Myotis ricketti, a species producing short frequency-modulated sonar pulses) with that from two species of Old World fruit bats lacking laryngeal echolocation(Rousettus leschenaultii, which uses a very rudimentary sonar system based on tongue-clicks, and Cynopterus sphinx, which lacks any echolocation). The most striking differences were seen in the ACC, which exhibited significant expression only in bats with laryngeal echolocation (Rhinolophus and Myotis), while in Tu, the signal labelling was more dense in Rousettus and Cynopterus, which commonly use mostly olfactory (and visual) cues instead of laryngeal echolocation to forage. No significant differences were observed between the species within each of the fruit bats and insectivorous bat groups. Therefore, this work not only confirmed our previous immunohistochemical results. We could also hypothesize that the expression of Foxp2in ACC may be related to laryngeal echolocation of insectivores, but in Tu may have some relationship with the food selection of fruit bats based on their olfactory neural system. Most importantly, our work could provide definitive morphofunctional locations for ensuing functional verification.3. Mammalian hibernation is characterized by prolonged bouts of torpor interspersed with brief periods of arousal. Adequate antioxidant defenses are needed to sustain the brain, an organ more susceptible to oxidative damage compared to most others in body because of its high oxygen utilization and the defense against high rates of oxyradical formation associated with massive oxygen-based thennogenesis during arousal. We characterized for the first time PrxⅢ, DJ-1and PrxⅠ, three peroxiredoxin immunohistochemical expression patterns in more than forty representative brain areas, to examine these protease distributions and the possible differentiation by comparison of the hibernating versus euthermic M ricketti. The results showed PrxIII, DJ-1and PrxⅠ were all expressed in both types of glia cells and neuron; moreover, we observed the whole distinct up-regulation of peroxiredoxins contributing to antioxidant defense during torpor. So this fully demonstrates that the family of peroxiredoxins most likely functions as the key brain antioxidant system of the hibernating mammals. Subsequently, through comparative analyses of the typical brain areas affected in ALS, AD and PD, such as primary motor cortex, hippocampus and substantia nigra between hibernators and non-hibernating model organisms, we further indicated the potential therapeutic value of the antioxidant neuroprotective mechanisms of hibernator. Lastly, considering the course of hibernation, we also hypothesized the remarkable antioxidant mechanism employed by hibernators is preconditioning, an endogenous neuroprotective mechanism in the brain based on peroxiredoxins and that this capacity may also have some relationship with the longevity of bats.更多还原