【作者】 陈坤；

【导师】 胡满；

【作者基本信息】 河北农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 本试验用c-fos法探讨光信息在母鸡中脑的传导通路,为深入了解光照对母鸡生产性能的影响机理打下基础。在养鸡生产中,光照是鸡赖以生存的环境中的重要因素之一;科学利用光照可以进一步提高鸡的生产性能,如:促进鸡的生长发育,调节性成熟,提高产蛋率、受精率、种蛋合格率等。禽类是最依赖视觉系统的脊椎动物。鸟类的视觉系统主要分为两个平行的视觉信息加工通路:离顶盖通路(thetectofugal pahway:视网膜—视顶盖—圆核—外纹体)和离丘脑通路(thethalamofugal pathway:视网膜—丘脑主视核和丘脑背外侧复合体—视厅);而对于禽类来说,离顶盖通路远比离丘脑通路重要的多。在鸡脑离顶盖视觉通路中,光刺激是否引起不同神经元的功能活动;哪些神经元参与某一特定光信息的传递,这些问题还有待于进一步研究。光线通过鸡的眼睛,刺激视网膜,经视神经叶的视神经途径传到丘脑下部或直接透过颅骨到达松果体及丘脑下部,下丘脑产生和分泌促性腺激素释放激素(Gonadotrophin Releasing Hormone,GnRH),再通过垂体门脉系统传至脑下垂体前叶,引起促卵泡激素(Follicle Stimulating Hormone,FSH)和促黄体激素(LutenizingHormone,LH)的分泌,LH和FSH协同促进卵泡成熟,促进卵巢分泌雌二醇。这些激素共同构成支配生殖机能的内分泌体系,影响生殖器官的发育;促使卵泡生长发育和排卵。但光信息在神经系统内部通过怎样的途径和方式对鸡下丘脑-垂体-性腺轴起调节作用,目前还不清楚。脑中神经元间的联系错综复杂,利用常规的神经束路追踪标记物方法不能对光信息通路进行完全的描绘,因为其一般不能跨神经元传递。但即早基因c-fos可通过跨突触活化作用诱导表达,在中枢神经系统接受内外刺激后,c-fos的表达产物Fos蛋白可作为单一神经元应答的分辨标记。c-fos原癌基因是一种即刻早基因(immediately early gene,IEG),耦连细胞外信息与细胞内靶基因的转录,被称为核内“第三信使”(third messenger);普遍存在于神经中枢系统(Central Nervous System,CNS),正常情况下高度保守,表达水平很低,难以检测,在受刺激的情况下可以迅速发生一过性表达。c-fos基因的表达可作为脑对包括光照在内的各种刺激起反应的标志物。疼痛刺激诱导c-fos基因在鸭脊髓的表达作为光诱导c-fos基因在母鸡中脑的表达的免疫组化阳性对照;从而对Fos蛋白的分布、形态有初步的认知。光诱导c-fos基因在母鸡中脑的表达试验:将鸡右眼遮光7天后接受20 1x的光照刺激1.5h,暗适应1.5h后灌流固定,取脑制作石蜡切片,采用免疫组化方法和Western Blot的方法检测c-fos基因在中脑的表达。结果显示:中脑内有大量Fos样免疫反应阳性神经元,主要见于左侧,分布于中脑SGC层、峡核大细胞部(Nucleusisthmi pars magnocellularis,Imc)和小细胞部(Nucleus isthmi pars parvocellularis,Ipc)、峡视核(Nucleus isthmo-opticus,ION)、半月核(Nucleus Semilunaris,SLu)、中脑外侧核背侧部(Nucleus mesencephalicus lateralis pars dorsalis,MLd)、红核(Nucleus raphes,RN)、滑车神经核(Nucleus nervitrochlearis,nⅣ)。中脑两侧峡核的Fos样免疫反应阳性神经元的细胞数密度及Fos样免疫反应阳性神经元所占神经核团细胞数量的百分比差异显著(P＜0.05)。但在未接受光刺激的脑区域内也有少量Fos样免疫反应阳性神经元。上述结果提示:光照信息主要通过视网膜经离顶盖通路传达到母鸡中脑,同时顶盖一峡核回路也起重要作用。非视觉感光系统(non-visual photosensitivesystem,NVPS)及离顶盖通路中光信息的交叉投射造成c-fos基因在未接受光刺激的脑区域中有少量的表达。更多还原

【Abstract】 The pathway of optical information in hen mesencephalon was investigated using c-fos method, to do some basic work for understanding the mechanism in the effect of light on egg-laying. In poultry production, light is one of the important environmental factors for maintaining the life of the animals. Scientific utilization of light can further enhance chicken’s productive performance, for example: promoting chicken’s growth and development, regulating sexual maturation, increasing egg production rate, fertility rate, hatching eggs eligible rate and so on. Poultry is one of vertebrate which is the most dependent on the visual system. Their visual system is mainly divided into two parallel processing pathways of visual information: the tectofugal pathway and the thalamofugal pathway. For poultry, the tectofugal pathway is more important than the thalamofugal pathway. In chicken’s tectofugal visual pathway, the problems that whether different light stimulation induce functional activities of different neurons or what are the neurons involved in the transmission of a specific light information have not be known yet.Light stimulates retina and the information is transmitted through optic nerve to hypothalamus or directly permeating skull arrives in conarium. Hypothalamus produces and secretes GnRH, which passing through pituitary portal system to anterior portion of hypophsis, inducing secretion of LH and FSH. LH cooperating with FSH facilitates follicular maturation, promoting ovary to secrete estradiol. These hormones jointly constitute endocrine system dominating procreation enginery and effect genital organs growth. But it is still unclear that in the nervous system, how the light information was adjusted.In encephalon, connection among neurons is labyrinthine. The conventional method to trace nervous pathway cannot diplay the integrated light pathway, because it generally cannot trace by trans-neuron. But immediate early gene-c-fos can be induced and expressed through trans-synaptic activation. When central nervous system received inside and outside stimulation, the Fos protein of expressive products of c-fos gene can be as a resolving tag of a single neuron response.c-fos is one of main member of immediate early gene in protooncogene. Coulping extracellular information in intracellular gene transcription, c-fos is described as "third messenger " in cell nucleus. Under normal circumstance, c-fos gene is commonly present in central nervous system, highly conserves and expresses at very low level, while in case of stimulated situation, a transient expression will be rapidly made. The expression of c-fos gene is a sign of response of encephalon receving various stimulations. The expression of c-fos gene induced by ache stimulation in ducks’ spinal cord is used as immunoreactive positive picture of the expression of c-fos gene induced by light stimulation in hen mesencephalon, primarily cognizing the distribution and morphology of Fos-like immunoreactive positive neuron in ducks’ spinal cord. The expression of c-fos gene was induced by light stimulation in hen mesecephalon: unilateral eyes of hens were kept dark for seven days. Then they accepted light stimulation of 20 lx for 1.5 hours and dark adaptation 1.5 hours. After perfusion with fixative, their brains were removed from the skulls and were made into continuous paraffin sections. Using immunohistochemical method and Western Blot method, expression of c-fos gene in mesencephalon was examined. The result showed that a number of Fos-like immunoreactive positive neurons were observed in mesencephalon, being mainly present in left stratum griseum centrale (SGC) of tectum, isthmi pars principals (Imc, Ipc), nucleus semilunaris (SLu), nucleus isthmo-opticus (ION), nucleus mesencephalicus lateralis (pars dorsalis, MLd), nucleus raphes (RN), and nucleus nervitrochlearis (nIV). And the Fos-like immunoreaction positive neurons were predominantly distributed in Imc and Ipc of the left mesencephalon, and the differentia of the cell density and the percentage chart of Fos-like immunoreactive positive neurons were distinct. But few Fos-like immunoreactive positive neurons were observed in encephalon regions without receiving light stimulation. The results suggested that though the optical information is mainly conveyed to mesencephalon through the tectofugal pathway, the tectoisthmi circus also play an important role in this course. The non-visual photosensitivesystem and light cross-projection in the tectofugal visual pathway cause the few expression of c-fos gene in encephalon regions without receiving light stimulation.更多还原