【作者】 林玲；

【导师】 章茜； 王志举；

【作者基本信息】 郑州大学 ， 生理学， 2010， 硕士

【摘要】 研究背景和目的：视皮层是视觉的最高级中枢,各种视觉信息经由视觉通路最终都要传导到视皮层,再经皮层的分析与综合产生视觉。目前,各种研究大鼠视皮层对不同闪光刺激的反应,主要集中在不同的刺激模式对视觉诱发电位的影响。闪光刺激对视觉中枢皮层神经元的场电位等自发电活动的影响,这方面的研究相对较少。皮层神经元的场电位反映的是大脑皮层锥体细胞的自发性电位活动,是大脑皮层锥体细胞同步化活动时产生的突触后电位或场电位的总和。闪光刺激对大鼠视皮层场电位产生明显影响,有报导提示增加刺激的强度和频率可以使视皮层场电位呈现强直后增强甚至长时程增强现象。不同颜色的闪光刺激对大鼠视皮层的影响也不同,据目前的研究,大鼠除了对红光刺激不太敏感,其他如白光及其他单色光刺激都能使大鼠视皮层场电位发生变化,但具体对哪种光刺激最敏感,不同的研究结果也不同,比较一致的观点是,大鼠对短波长的光如紫外光等刺激比较敏感,可能与光量子的强度较大有关,也与大鼠视网膜感光细胞类型有关。单色光刺激对大鼠视皮层电活动的影响,据有关文献报道,主要是通过皮层与丘脑或丘脑与皮层之间形成的正反馈环路有关,也有研究表明在大鼠视皮层V1区存在对颜色视觉特异性敏感的神经细胞,而且现在通过功能性的磁共振技术证实这些细胞具有对不同颜色刺激的分辨能力。鼠科动物视锥细胞的感光蛋白主要有两种类型,M-opsin和UV(S)-opsin,也有研究提示有些视锥细胞既表达M-opsin,也表达UV-opsin, M-opsin几乎在所有的哺乳动物视网膜中都存在,其与动物的生存、繁衍都具有密切的关系,S-opsin是决定大鼠是否具有颜色视觉的关键感光色素,但大鼠是否具有功能性的S-opsin还有待进一步证实,大鼠的行为学测试结果也为其可能存在一定形式的颜色视觉提供了证据,但大鼠颜色视觉的具体情况目前并不是很清楚。本实验拟采用白色及不同单色光的刺激,探讨色光刺激对大鼠视皮层脑电活动的影响以及可能的强直后增强现象,进一步加深对中枢神经系统神经元可塑性的认识,为进一步了解大鼠是否具有颜色视觉以及单色光刺激对初级视皮层电活动的影响的机制,采用免疫组化方法研究大鼠视网膜视杆细胞和视锥细胞感光色素以及视皮层感光色素的表达,为脑认知的研究奠定一定的理论基础。言法：1电生理记录：健康成年SD大鼠10只,体重180g-220g,雌雄不拘。水合氯醛腹腔注射麻醉,暴露颅骨,参照大鼠脑解剖图谱定位初级视皮层V1区(前囟向后7.0mm,左旁2.0mm),分别将记录电极置于V1区,参考电极E1(前囟向前2.0mm,右旁2.0mm)固定于额叶,牙托粉固定。于术后第5、7、9 d行脑电信号采集。将已植入电极大鼠置于有机玻璃鼠箱中,在鼠箱侧壁安装发光二极管,分别给予大鼠白、红、绿LED闪光刺激。每次记录约2h,描记前经30min暗适应,之后依次给予白、红、绿闪光刺激,不同色光刺激转换之间,间歇30min,并同时经多道生理记录仪记录Ⅵ脑电波的变化。分别测定实验大鼠术后第5、7、9d给予白、红、绿光刺激前后各6s时间段,大鼠在清醒和探究行为状态时V1区皮层场电位的幅值和面积。2免疫组化2.1大鼠视网膜感光蛋白Rhod-opsin和S-opsin的表达：2.1.1将成年SD大鼠麻醉,迅速取出眼球,行连续冰冻切片,对视网膜进行免疫荧光双标：一抗为鼠多克隆抗Rhod-opsin抗体(1:400)+兔单克隆抗S-opsin抗体(1:400),二抗为FITC-羊抗鼠IgG(1:250)和TEXRED-羊抗兔IgG(1:250)2.1.2将成年SD大鼠麻醉,4%多聚甲醛心脏灌注迅速取出眼球,石蜡切片,一抗为鼠多克隆抗Rhod-opsin抗体(1:400)+兔单克隆抗S-opsin抗体(1:400),二抗为羊抗鼠IgG(1:250)和羊抗兔IgG(1:250)。2.2大鼠初级视皮层感光蛋白S-opsin的表达：2.2.1将成年SD大鼠麻醉,断头取脑,对初级视皮层行连续冰冻切片,免疫荧光抗体技术鉴定视皮层感光蛋白S-opsin:一抗为兔单克隆抗S-opsin抗体(1：400),4℃孵育过夜,二抗为TEXRED羊抗兔IgG(1：250)。2.2.2将成年SD大鼠麻醉,4%多聚甲醛心脏灌注迅速取出脑组织,石蜡切片,一为抗兔单克隆抗S-opsin抗体(1：400),二抗为羊抗兔IgG(1：250)。3结果：3.1电生理实验结果3.1.1场电位随不同色光刺激天数的变化：结果显示,4次重复测量的数据之间存在高度的相关性(P<0.01),组内(校正值)和组间的方差分析结果显示,时间因素、色光因素以及时间与色光的交互作用均有显著性统计学意义(P<0.05)。说明幅值和面积有随重复刺激天数变化的趋势,并且这种趋势随着色光的不同而不同。3.1.2每一实验日不同色光之间引起场电位的两两比较实验日第5天刺激前与刺激后相比较,各种色光引起初级视皮层V1区脑电波场电位幅值和面积均未见显著性差异；实验日第7天给于相同色光刺激,白、绿色光引起V1区场电位的幅值和面积均显著性强于刺激前(白光,P<0.01；绿光,P<0.05),白光同时明显强于红光(P<0.05)；实验日第9天再次给于相同色光刺激,白、绿色光引起V1区场电位的幅值和面积均极显著性强于刺激前(P<0.01),且白绿色光均明显强于红光(P<0.01)。红光在各实验日刺激前后比较均未见显著性差别。3.1.3每种色光不同实验日间场电位的两两比较3.1.3.1对白光分析实验日第7天和第9天V1区场电位的的幅值和面积均显著强于刺激前和第5天(第7、9天VS刺激前P<0.01,第7、9天VS第5天P<0.05),余组间未见显著性差异。3.1.3.2对绿光分析实验日第7天和第9天的V1区场电位的幅值和面积均显著强于刺激前(幅值,第7天VS刺激前P<0.05,第9天VS刺激前P<0.01,面积第7、9天VS刺激前P<0.01),余组间未见显著性差异。3.1.3.3对红光分析不同实验日间的幅值和面积均未见显著性差异(P>0.05)。3.2免疫组化实验结果3.2.1大鼠视网膜感光蛋白Rhod-opsin和S-opsin表达阳性。3.2.2大鼠初级视皮层S-opsin表达阳性。结论：1.白、绿色闪光刺激能显著增强大鼠初级视皮层的电活动；呈现敏感化和强直后增强现象,大鼠初级视皮层神经元具有一定程度的可塑性。2.红色闪光刺激对大鼠初级视皮层电活动无显著影响。3.不同色光闪光刺激致大鼠初级视皮层电活动的变化,白、绿之间无显著性差异。4.大鼠视网膜有视杆细胞感光蛋白及S型视锥细胞感光蛋白的表达,大鼠初级视皮层有S型感光蛋白的表达。5.大鼠可能具有某种形式的颜色视觉。更多还原

【Abstract】 Background and Objective:Visual cortex is the highest pivot of vision. All sorts of visual information which transmit through visual path to visual cortex, causing the variation of potential in the neurons of visual cortex, that eventually makes visual sense. At present, among the research about the reaction of rat’s visual cortex to different flashing stimulations, existing ones mainly focus on the influence to visual induced potential by kinds of stimulate patterns. There is relatively little research in this respect, which concerning the impact on the spontaneous electrical activity by flashing stimulation to the field potential of neurons in visual pivot. The field potential of cortical neurons which reflect the spontaneous electrical activity of pyramidal cell in cerebral cortex, is the sum of PSP or field potential caused in the process of the synchronous activities by pyramidal cell in cerebral cortex. Flashing stimulations made by different colors has multifarious influences on rats’visual cortex. According to the present research, except for the insensitivity for red color, the rats can react to other light such as white light or all other monochromatic light and make the field potential of cortical neurons change. But to what kind of color the rat is most sensitive, there is no final conclusion so far. Most relevant experts think that the rat is more sensitive to the light of short wave such as UV-light. This phenomenon is possibly because of the mass intensity of optical photon, and partly has some relation to the type of the photoreceptor cell in the retina of the rats.Flashing stimulations have also made distinct influence on the field potential of the rats’visual cortex. It is presented by some report that increasing the intensity and frequency of the stimulation can result in posttetanic potentiation, PTP and even long-term potentiation, LTP.It is reported that the influence on the electrical activity of rats’visual cortex by the monochromatic light, is mainly by how the neural circuitry is altered, after the initial flash, to receive succeeding flashes in the photic stimulus train. This likely reflects alterations within recurrent feedback loops within cortex and thalamus and/or from cortex to thalamus, rather than a change in the direct excitatory connections in the ascending retino-geniculo-cortical pathway.But it is unclear that the specific mechanism of the field potentiation change induce by the different color photographic stimulation as well as the rats’color vision.The opsins of the murine cone cell can be mainly divided into two types, one of which is called M-opsin and the other is called UV-opsin. There are also researches which indicate that some of the rats’cone cell can express not only M-opsin, but also UV-opsin. almost all mammalian retina are present M-opsin, S-opsin is to determine whether rats with key photosensitive pigment color vision, The consequence of the rats’behavioral testing may well provide evidence for the existence of color vision in certain forms.This experiment is planed to using white light and other monochromatic light, which intend to discuss the influence of brain electrical activity of the rats’visual cortex and even possible long-term potentiation, LTP by the light stimulations, will enhance the recognization to the plasticity of the central nervous system, and eventually lay the first stone for the research to cerebral cognitive function. In order to understanding the influence of the monochromatic light to the electrical activity of rats’primary visual cortex. Methods:1 Electrophysiological experiments:10 healthy adult SD rats, weighing 180g-220g, either male or female. Intraperitoneal injection of chloral hydrate anesthesia, exposing the skull, brain anatomical mapping in reference to the primary visual cortex Vl area (6.8-7.0mm posterior to lambda,2.0mm lateral), recording electrodes were placed in the Vl area, reference electrode El (2.0mm anterior to lambda,2.0mm lateral) fixed on the frontal lobe, both the E2 (-2.0mm anterior to lambda,2.0mm lateral) as a reference, denture powder fixed.5,7,9 d after the first line in the EEG signal acquisition. Rats with implanted electrodes have been placed in the plexiglass box rat, mouse box in the wall to install light-emitting diode, respectively, rats were given white, red, green LED flash stimulation. Each record of about 2h, tracings before dark adaptation after 30min, and then turn to give white, red, green flash stimulation, stimulation of conversion between different color light, intermittent 30min, and also by the multi-channel physiological recorder records brain wave changes in District VI. Rats were measured after the first 5,7,9 d to give white, red, green,6s time before and after stimulation, and exploratory behavior in rats in the awake state..2 Immunohistochemistry2.1 Retinal photoreceptor protein Rhod-opsin and S-opsin expression:2.1.1 The adult SD rats were anesthetized, the eye quickly removed, frozen sections of continuous lines of double immunofluorescence retina:The first antibody are the mouse polyclonal anti-Rhod-opsin antibody (1:400) and rabbit anti-S-opsin monoclonal antibody (1:400), the secondary antibody are FITC-goat anti-mouse IgG (1:250) and TEXRED-goat anti-rabbit IgG (1:250).2.2.2 The adult SD rats were anesthetized with 4% paraformaldehyde perfusion eyeballs quickly removed, paraffin sections, The first antibody is the mouse polyclonal anti-Rhod-opsin antibody (1:400) and rabbit anti-S-opsin monoclonal antibody (1:400), the second antibody was goat anti-mouse IgG (1:250) and goat anti-rabbit IgG (1:250).2.2 The primary visual cortex photoreceptor protein S-opsin expression: 2.2.1 The adult SD rats were anesthetized, brains were removed, the primary visual cortex of a continuous line of frozen sections and immunofluorescence antibody technique identified the visual cortex of light-sensitive protein S-opsin:The first antibody is rabbit anti-S-opsin antibody (1:400),4℃overnight incubation, the secondary antibody is TEXRED-goat anti-rabbit IgG (1:250).2.2.2 The adult SD rats were anesthetized with 4% paraformaldehyde perfused brains were removed quickly, paraffin sections, The first antibody is anti-rabbit anti-S-opsin monoclonal antibody (1:400), the secondary antibody is goat anti-rabbit IgG (1:250.)Results:1. Electrophysiological experiments1.1 Field potential stimulation with different shade of change in the number of daysBrain wave amplitude and area of the test showed high correlation between that the 4 repeated measurement data, (P<0.01), group (correction) and between groups Analysis of variance showed that the time factor, and shade factors were significant interaction was significant (P<0.05), the amplitude and area of days shows the trends with repetitive stimulation, and this trend with the color vary light.1.2 Each experiment on field potential induced between the different shade of pairwise comparisonEach experiment on field potential induced between the different shade of pairwise comparison AT ay 5,compared before and after stimulation, a variety of shade caused by primary visual cortex area V1 brain wave amplitude and area of field potentials were no significant differences; at day 7, to stimulate the same shade, white, Green light field caused by the amplitude V1 area and area were significantly stronger than before (white, P<0.01; green, P<0.05), while white light was stronger than the red(P<0.05); at day 9,once again stimulated in the same shade, white, and green light cause the amplitude of field potentials V1 area and area were significantly stronger than before stimulation (P<0.01), the white and green light were significantly stronger than the red (P<0.01). Stimulation in all experiments at red light before and after showed no significant difference.1.3 The comparison of each shade of the field potentials at each day1.3.1 To white light At day 9, Vl field potential amplitude and area were significantly stronger than before stimulation and day 5, (days 7,9 VS before stimulation P<0.01, days 7,9 VS day 5, P<0.05), there was no significant differences between other groups.1.3.2 To green light At day 7and 9, Vl field potential amplitude and area were significantly stronger than before stimulation, (days 7 VS before stimulation P<0.05, days 9 VS before stimulation, P<0.01,and the area,days 7 and 9 VS before stimulation, P<0.01), there was no significant differences between other group.1.3.3 To red light There was no significant differences between all groups at each experiment days.2. Immunohistochemistry results2.1 Retinal photoreceptor protein Rhod-opsin and S-opsin expression is positive.2.2 The expression of S-opsin in Primary visual cortex in is positive.Summary:1 White and green flashing stimulation can enhance the electrical activity in rats’ primary visual cortex, Suggesting sensitization and post-tetanic potentiation of primary visual cortex.2 There is no obvious effect on the electrical activity in the rats primary visual cortex electrical activity to red flashing stimilation.3 There is no significant difference between white and green flash.4 Expression Rhod-opsin and S-opsin in the rats’retinal, and expression S-opsin in rats’ primary visval cortex5 Rat may has a form of color vision.更多还原