【作者】 赵志富；

【导师】 暴学祥；

【作者基本信息】 东北师范大学 ， 动物学， 2008， 硕士

【摘要】 柞蚕蛾(Antheraea pernyi)个体较大,脑结构相对完整,各种神经细胞分化明显,易饲养,是研究脑神经生物学较好的昆虫模型。5-HT与DA作为单胺类神经递质在昆虫脑中起着重要的调节作用,两者也与多种脊椎动物神经类疾病有着密切的联系。单胺类神经递质的合成、转运与代谢越来越成为人们研究的热点。昆虫脑内特异的5-HT合成限速酶TPH的发现,改变了以往人们对昆虫脑内芳香族氨基酸羟化酶的分化和对单胺类神经递质合成途径的看法,为进一步研究昆虫脑内单胺类神经递质的合成问题开辟了新的研究思路。相对于荧光双标等技术,树脂石蜡(CP)组织包埋并制作连续相邻切片的技术可以更直观、更明显的将两种功能神经元的分布及可能存在的共定位显示出来。其制作过程与传统石蜡切片相比,具有明显的优越性。本文采用CP组织包埋切片技术,制备柞蚕蛾脑组织相邻切片,结合免疫组织化学链霉菌抗生物素蛋白-过氧化物酶(SP)双染法,在同一脑组织内,对5-HT和DA及其限速酶TPH和TH的定位进行研究。免疫组织化学结果显示,在柞蚕蛾的视叶与侧前脑间、前脑背方、中脑及咽下神经节存在TPH免疫阳性反应细胞群。相同实验条件下的5-HT免疫组织化学实验表明,TPH免疫阳性反应细胞群的分布模式与5-HT极度吻合。该结果有力的证明了昆虫脑中TPH的客观存在,并说明至少在节肢动物中芳香族氨基酸羟化酶已经有了TPH的分化。与其他实验昆虫相比,柞蚕蛾脑内视叶与侧前脑间的TPH阳性反应神经元数目最多,它们大多数为切向神经元。前脑背方的TPH阳性反应神经元通过突触与视叶及蕈形体发生联系。柞蚕蛾中脑存在一对TPH免疫阳性反应细胞,并与嗅觉神经发生联系。柞蚕蛾的后脑及咽下神经节有TPH免疫阳性反应细胞向后脑发出树突状分支。柞蚕蛾的咽下神经节腹前方有一对神经元,其胞体的TPH免疫组织化学阳性反应强烈,而5-HT阳性反应极弱。在相同控制实验条件下,我们做了DA与TH的免疫组织化学研究,但并未检测出DA或TH阳性神经元。通过对购置抗体的免疫蛋白印迹的研究,我们认为这可能与所用抗体的特异性或实验过程中某些环境因素影响了这种神经递质的检测标记。更多还原

【Abstract】 Antheraea pernyi is a classic species for studying insect brain nerves system because of its large size, relatively intact structure of brain and obvious cytodifferentiation. 5-HT and DA, the amine neurotransmitters, function as very important mediators in the insect brain. Meanwhile, in the vertebrate, they are closely correlated with nerves diseases, and their synthesis, transport and metabolism have been more and more concerned. The discovery of specific rate-limiting enzyme TPH responsible for synthesis of 5-HT made people look insight into the evolution of AAAH and synthesis of amine neurotransmitter, providing a novel pathway for studying amine neurotransmitter in the insect brain. The Colophony-Paraffin embedded section combining the adjacent section technique, can provide the view of the distribution of two kinds of neurons more obviously. In this paper, anti-TPH and anti-TH antisera were applied on the brain adjacent section of Antheraea pernyi, using the Streptavidin-Peroxidase immunohistochemical method and the Colophony-Paraffin embedded section technique. Results revealed that, there were TPH positive cell bodies, which located between the optic lobe and lateral protocerebrum, dosal protocerebrum, deutocerebrum and suboesophageal ganglion respectively. the pattern of TPH immunoreactivity of cell body is extreme similar to that of 5-HT. It implies that the PAH-TPH duplication may have occurred before the emergence of arthropods. The number of TPH positive neurons in the optic lobe and lateral protocerebrum is the largest among other insects we studied, and most of them are tangential neurons. TPH immunoreactive neurons in the dorsal protocerebrum interconnected with optic lobes and mushroom bodies. There was only one pair of TPH immunoreactive neurons in deutocerebrum,which interconnected with olfactory nerves. TPH immunoreactive neurons and fibers were present in tritocerebrum and suboesophageal ganglion, in which there was a group of neurons located in interior tritocerebrum that invaded the tritocerebrum by their dendrites. There were two giant isomorphic neurons located antero-ventral to the SOG neuropil in each hemisphere displayed strong TPH immunoreactivity, however, the somata of these two neurons were weakly labeled by anti-5-HT antiserum.. With regard to the immunohistochemical studies of DA and TH, we had not found the similar pattern to that of 5-HT and TPH. We had identified the purchased antibody with western blotting and analyzed the protocol of the experiments, raising the possibility that specificity of the antibody and some factors in the experiments affected the labeling of this neurotransmitter and its rate-limiting enzyme.更多还原