家犬（Canis familiaris）、狼（Canis lupus）嗅觉系统研究

【作者】 韦钦国；

【导师】 张洪海；

【作者基本信息】 曲阜师范大学 ， 动物学， 2008， 硕士

【摘要】 家犬(canis familiaris)是一种家养犬科动物,经过人类驯化,大约在15000年前,由狼进化而来。狼(Canis lupus),家犬的祖先,在行为和生理方面都表现出对野外生活的适应性,它拥有不容侵犯的领域。本研究用组织学和免疫组织化学方法,对不同性别的幼年和成年家犬的嗅球的组织学结构以及这些结构中c-Fos蛋白的表达进行研究,以探讨家犬嗅觉系统的发育过程和嗅觉系统的结构和功能在年龄、性别之间的差异。本研究还利用分子生物学方法,对家犬和狼的嗅觉受体基因进行比较研究,以探讨驯化对家犬嗅觉受体基因的影响,并通过与其它物种的比较,初步探讨狼和家犬嗅觉灵敏的原因。研究结果表明:1嗅球呈卵圆形,位于筛骨的后方,脑的最前部。嗅球的结构从外到内可以分为:嗅神经层(ONL,olfactory nerve layer)、嗅小球层(GL,glomeruli layer)、外网丛层(EPL,externalplexiform layer)、僧帽细胞层(ML,mitral cell layer)、内网丛层(IPL,internal plexiform layer)和颗粒细胞层(GRL,granule cell layer)。嗅球接受嗅觉上皮神经元通过轴突传来的嗅觉信号,并与这些轴突在嗅小球处发生突触联系,实现信息的传递。2在幼年家犬中,嗅球的基本结构已经出现,已经有很明显的分层结构。说明幼年家犬已经具备基本的嗅觉能力。统计学分析表明,幼年家犬嗅球各层的宽度未出现性二型分化(P＞0.05);嗅小球层的细胞密度存在极显著的性别差异(P＜0.01),幼年雌性家犬该层的细胞密度明显偏高,这可能表明雌性家犬嗅小球层的结构发育较快。其余各层的细胞密度,在雌雄幼年家犬之间,未发现显著的差异(P＞0.05)。一些层次的细胞密度由高到低的顺序为GRL＞GL＞ONL＞EPL。3在成年家犬中,嗅球各个层次的宽度和细胞密度(除僧帽细胞层)也没有显著的性别差异(P＞0.05)。但雌性家犬单位视野内僧帽细胞的数量显著高于雄牲家犬(P＜0.05),僧帽细胞的形态在雌雄动物之间也有很大的不同,雌性家犬的僧帽细胞胞体呈帽状,突起较为明显,胞质染色较深,雄性家犬僧帽细胞体约呈卵圆形,胞体颜色较浅。僧帽细胞是嗅球中的重要输出性神经元,也是嗅球中最大的细胞,它在雌雄动物之间的显著差异,可能会造成雌雄动物之间嗅觉能力的差异。各层次的细胞密度高低顺序与幼年家犬的一致。4幼年和成年家犬嗅球结构的比较发现,成年家犬的嗅球体积、重量和各个层次的厚度相对于幼年家犬都显著的增加,细胞密度随着年龄的增长明显降低,两者之间的差异都达到了显著或极显著的水平。这表明,幼年到成年的过程中,嗅球的结构也处在不断的发育和完善过程之中。5免疫组化结果显示,幼年家犬嗅球的各个层次中均有c-Fos蛋白的表达,嗅小球层的表达率存在明显的性别差异(P＜0.05),其它各层的表达率在雌雄动物之间无明显差异(P＞0.05)。阳性细胞主要集中在颗粒细胞层,僧帽细胞层和嗅小球层。表达率由高到低依次为ML＞GRL＞EPL＞GL＞ONL。6成年家犬嗅球的各个层次中也都有较多的c-Fos蛋白的表达,各层的表达率也没有显著的性别差异。阳性细胞的分布情况与幼年家犬的类似,在颗粒细胞层发现层状分布。表达率顺序为ML＞EPL＞ONL＞GL＞GRL。7成年家犬和幼年家犬相比,嗅球中阳性细胞染色更加清晰,数目也更多,表达率显著增高。而幼年家犬的阳性神经元的颜色较浅,模糊不清晰。这说明,在幼年时期,嗅球的神经元主要处在发生和发育过程之中,凋亡的事件相对较少,记忆能力相对较差。到了成年阶段,家犬嗅球神经元的活动变得更加活跃,嗅觉系统也发展的更为完善,c-Fos的大量表达,可能说明成年家犬嗅球中的细胞具有更强的学习记忆功能。8分子生物学实验研究表明,家犬的嗅觉受体基因中假基因的比例比狼的偏高,但没有显著的差异(X~2=1.388,P=0.239＞0.05)。这说明,虽然家犬是家养动物,通过人的驯化,由狼进化而来,但驯养并未导致明显的嗅觉受体基因退化现象,这可能与驯化的目的有关。与其它一些物种相比,家犬和狼的假基因比例明显偏低,说明家犬和狼具有更为灵敏的嗅觉。更多还原

【Abstract】 The watchdog (canis familiaris), the world’s oldest domesticate, is a very close relative of the grey wolf (Canis lupus) and the two species diverged anywhere from 145 00 to 15 000 years ago. The wolf, ancestor of watchdog, whose behavior and physiological features are adapt to field lives, has their own domains that are not allowed to be invaded. In order to probe into the developmental course of watchdogs’ olfactory system and their structure difference between watchdogs of different age and different sex, the methods of histological and immunohistochemical were used in this paper to detect the olfactory bulb’s(OB) histological structure and the expression of c-Fos in its each layer in watchdogs of different age and different sex. We also use molecular methods to study olfactory receptor genes of watchdogs and wolves comparatively for the purpose of exploring the influence of domestication on watchdogs’ olfactory receptor genes. At the same time, we compared the composition of olfactory receptor genes of watchdogs and wolves with other animals’ in the interest of discussing the reasons for the powerful sense of smell held by canine. The results of our experiments showed as follows:1. The watchdogs’ OB, which lies behind the ethmoid and at the frontal part of the brain, was oval. Its main structures include ONL, GL, EPL, ML, IPL and GRL. And it accepts odorant signals transmitted by olfactory receptor neurons whose axons are bundled in groups to penetrate the ethmoidal cribriform plate of bone, reaching the OB of the brain where they converge to terminate with post-synaptic cells to form synaptic structures called glomeruli.2. The appearance of primary structures in the OB of juvenile watchdogs demonstrates that they already possess the potential ability for distinguishing odorants. The results of statistical analysis told us that there was no significant difference on the width of OB’s each layer and the cells’ density of ONL, EPL and GRL between juvenile watchdogs of different sex (P>0.05). However, the difference on the density of GL’s cells reached the remarkably significant level (P>0.01). The apparent higher density of cells in GL of female juvenile demonstrated their faster development of this layer. On the other hand, the rank of cells’ density is GRL>GL>ONL>EPL.3. There was also no significant difference on the width of OB’s each layer and the cells’ density of ONL, EPL and GRL between adult watchdogs of different sex (P>0.05). Nevertheless, the difference of mitral cells’ number and their morphology between adult males and adult females was apparent (P<0.05). Just like its name, the morphology of females’ mitral cells is mitral, and it also has evident prominency and heavy color in nucleolus, whereas the color of the oval mitral cells of males is light. Mitral cells are the main efferent cells in the OB, and they are also the largest cells in the OB. The difference mentioned above might be one of the reasons for the difference of smell sensitivity between males and females as the result of the importance of mitral cells in olfactory system. In addition, the cells’ density rank is the same as that in juveniles’ OB.4. The comparison of OB’s structure between juveniles and adults showed that the OB’s weight, volume and width increased evidently with the growth of age. However, the density of cells of its each layer decreased significantly as the watchdogs grow old. This change suggested that the OB was developing during the course of watchdogs’ growth.5. We found c-Fos immunoreactivity neurons (c-Fos-ir) in every layer of juveniles’ OB according to the results got from the immunohistochemical experiments. The difference of the expression ratio of c-Fos in GL between female juvenile and males was at the significant level (P<0.05), but there was no significant difference on the expression ratio of c-Fos in OB’ other layers between juveniles of different sex (P>0.05). c-Fos-ir neurons mainly distributed in GRL, ML and GL, and the rank of its expression ratio is ML>GRL>EPL>GL>ONL.6. We also found many c-Fos immunoreactivity neurons in every layer of adults’ OB, and there was no significant difference on the expression ratio of c-Fos in OB’ each layer between adult watchdogs of different sex (P>0.05). c-Fos-ir neurons mainly distributed.in GRL, ML and GL, and the rank of its expression ratio is ML>EPL>ONL>GL>GRL.7. The c-Fos-ir neurons in OB of adults were much clearer, their color was much heavier and their number was much larger than that in juveniles’ OB. All of these implied that cells in juveniles’ OB were during the course of development and cytogenesis, and the apoptosis event was fewer accordingly; the OB’s cells of juveniles might have a weak ability for memorizing odorant information. On the other hand, cells in OB of adult watchdogs were much more active and they might have stronger memory abilities than that of juveniles.8. The results of molecular experiments illuminated that the percentage of pesudogenes in watchdogs’ olfactory receptor gene repertoire was larger than that in wolves, but the difference did not reach the significant level (X2=1.388, P=0.239>0.05). This indicated that domestication did not reduce the number of functional olfactory receptor genes in watchdogs evidently as the result of the purpose of its domestication. We found that the proportion of pseudogenes in olfactory receptor gene repertoire of watchdogs and wolves were much smaller than that in other species, this demonstrated that canine have a stronger ability for discerning odorants than that of other animals.更多还原