胆碱能神经元调控海马齿状回区新生神经元的作用研究

发布时间：2018-05-28 02:39

  本文选题：胆碱能神经元 + 新生神经元　； 参考：《华中科技大学》2016年博士论文

【摘要】：[背景]上世纪90年代以前,研究人员普遍认为在成年哺乳动物脑中的神经元不能再生。因此,人们认为在人的一生中,任何由于其它因素(如：中风、头部创伤、氧化应激、神经退行性疾病、衰老等)导致的神经元死亡,都将导致其功能的永久丧失。然而,早在上世纪60年代,阿尔特曼(Altman)和其同事就质疑了这一观点的可靠性。他们报道,在成年大鼠正常脑组织的至少两个区域(海马颗粒下层(the subgranuar zone,SGZ)和侧脑室下区(the subventricular zone, SVZ))内,新生神经元能持续不断地产生。之后,海马颗粒下层的新生神经元逐渐迁移到齿状回的颗粒层内,侧脑室下区的新生神经元则迁移到嗅球。由于受出生后的哺乳动物大脑中不存在神经元再生这种观点的影响,使得阿尔特曼和戴斯(Das) (1965)的发现三十多年后才被大家广泛接受。哺乳动物的多能神经干细胞位于SGZ和SVZ。这些干细胞首先不对称地分裂为一个子代祖细胞和一个子代干细胞。然后前者能再不对称地分裂为可分化成星形胶质细胞或神经元的子代细胞,并且祖细胞保持有多次分裂的能力。对这些新生细胞生长、存活和分化的研究,常常涉及到外源性给予胸苷类似物或溴脱氧尿甙(Brdu)这些可标记分裂细胞的物质。成年哺乳动物大脑中的神经元再生过程常受到许多内在和外在因素的强烈影响。局部微环境(“神经源性壁龛”或“干细胞壁龛”)中的内源性外在因素包括：神经前体细胞、周边成熟细胞、细胞间相互作用、细胞纤毛、细胞分泌物和神经递质。现在的研究认为在SGZ和SVZ的微环境中,而不是大脑其它区域,存在一些特定的调控新生神经元生长发育的因素。一项权威的研究表明在成年小鼠海马中的星形胶质细胞促进体外成年源性海马祖细胞的神经分化。乙酰胆碱(acetvlcholine. ACh)作为一种重要的神经递质,它是被人类最早发现,并在生物体内参与很多重要功能的递质,如：调控学习与记忆、情绪和睡眠等。虽然胆碱能神经元胞体只分布在大脑中一些特定的核团和区域,但其轴突几乎支配整个大脑组织。例如,海马中虽然几乎没有胆碱能神经元胞体的分布,但其间却遍布来自基底前脑的胆碱能神经纤维。许多研究表明GABA能神经元、谷氨酸能神经元和多巴胺能神经元对海马齿状回区的新生神经元具有重要的调控作用,但作为重要神经递质(乙酰胆碱)来源的胆碱能神经元却鲜有报道。[目的]本研究的目的主要包括三部分：1.应用转基因和光遗传手段特异性抑制或激活基底前脑胆碱能神经元,观察胆碱能神经元对海马齿状回区新生神经元的调控作用2.运用电生理、病毒干扰和免疫组织化学等技术探讨上述调控作用的具体途径。3.完美呈现小鼠大脑中胆碱能神经元分布的高清三维图像。[方法]特异性抑制胆碱能神经元对海马齿状回区新生神经元数目的调控作用：为了特异性抑制小鼠大脑内的胆碱能神经元,我们首先将实验室自主构建的Kir2.1工具鼠(loxp-stop-loxp-Kir2.1-tdTomato)与在胆碱能神经元中表达Cre重组酶的(ChAT-Cre)转基因小鼠杂交,经多次扩增杂交和PCR鉴定后得到两种转基因小鼠(ChAT+/Kir2.1+,ChAT+/Kir2.1-)。将这两种转基因小鼠再分为注射他莫昔芬(Tamoxifen)组和注射玉米油(Oil)组,共计四组：ChAT+/Kir2.1+/Tamoxifen,ChAT+/Kir2.1+/Oil,ChAT+/Kir2.1-/Tamoxifen, ChAT+/Kir2.1-/oil。待Kir2.1充分表达后,给各组小鼠腹腔注射Brdu.然后分别标记在第1天(d)、4d、7d、14d、28d和42d时海马齿状回区(DG)的Brdu并计数。最后统计在各个时间点各组间Brdu数量的差异。特异性激活基底前脑区胆碱能神经元并观察DG区新生神经元数目的恢复情况：首先将ChAT+/Kir2.1+小鼠和ChR2小鼠杂交数代,得到两种转基因小鼠(ChAT+/Kir2.1+ChR2+,ChAT+/Kir2.1+ChR2-)。选取一些雄性后代进行Tamoxi fen腹腔注射,然后在其基底前脑部埋置光纤。待Kir2.1和ChR2充分表达后,给各组小鼠腹腔注射Brdu。从14d开始对小鼠给予光激活处理(实验小鼠分为两组：ChAT+/Kir2.1+ChR2+/给光组、ChAT+/Kir2.1+ChR2-/给光组),然后分别标记28d和42d时DG区的Brdu并计数。最后统计在各个时间点各组间Brdu数量的差异。观察新生神经元上胆碱能受体的表达情况：选取一批1月龄的雄性C57BL/6小鼠,在其DG区注射逆转录病毒(RV-GFP)。分别于4d、7d、14d、21d、28d和42d时结合胆碱能受体拮抗剂进行电生理检测,以及对受体类型进行免疫组化标记确证。阻断DG区新生神经元上胆碱能受体对光激活模型小鼠中新生神经元数目的影响：基于上述光激活模型小鼠,通过注射逆转录病毒特异性阻断新生神经元上胆碱能受体,标记上述两个时间点(28d和42d)的Brdu并计数,然后进行统计分析。小鼠大脑中胆碱能神经元分布的全脑三维重构：选取一只3月龄的雄性C57BL/6小鼠,对其脑组织进行矢状面的连续切片(16μm)。对所有矢状面脑片进行ChAT免疫组化染色,然后对脑片进行二维的全脑扫图。对所有拍摄好的大图进行角度和位置的校准,最后将校准好的图片进行三维合成,并导出相应的图片和视频。此外,针对相关区域,首先用PhotoShop软件在校准好的图片上进行截图,然后将这些裁剪出的图片进行三维合成并导出数据。[结果]在观察胆碱能神经元对DG区新生神经元调控的研究中,我们发现在胆碱能抑制组中,DG区Brdu的数目在1d、4d、7d和14d时和其它组间没有明显差异,而在28d和42d时则显著降低。并且在给予光激活治疗后,这两个时间点的Brdu数目则趋向复原。为了进一步探讨是哪种(或哪类)胆碱能受体参与了这种调控作用,我们发现第4d、7d的新生神经元对ACh的刺激均没有反应,而在14d、21d和28d时对ACh的刺激有反应,且呈明显的递增趋势,并且这种反应只能被M型或M1型乙酰胆碱受体拮抗剂阻断;同时,我们的免疫组织化学结果也显示,只有14d、21d和28d时的新生神经元能与M1受体共标。随后我们通过特异性阻断新生神经元上的M1型受体,发现上述光激活的治疗作用明显减弱。我们首次应用免疫组织化学技术成功构建了小鼠大脑中胆碱能神经元分布的三维图像。在此三维图像中,胆碱能神经元分布的各个核团清晰可见,其中：尾壳核(the caudate putamen)中有约11000个胆碱能神经元、基底前脑(the basal forebrain)中约12000个胆碱能神经元、脑干脚桥核与脑干侧被盖核(the pedunculopontine nucleus and the laterodorsal tegmental nucleus)中约1200个胆碱能神经元、脑干面神经核(the facial nucleus of the brainstem)中约2200个胆碱能神经元、脑干背运动核(the dorsal motor nucleus of the brainstem)中约300个胆碱能神经元,并且全脑中(不包含皮质)胆碱能神经元的数目不超过40000个。[结论]基底前脑区的胆碱能神经元通过M1受体调控新生神经元的存活,而对其发生和分裂则没有明显影响,这可能为今后治疗记忆缺陷提供理论依据。
[Abstract]:[background] before the 90s of last century, researchers generally believed that neurons in the brain of adult mammals could not be regenerated. Therefore, it is believed that any neuron death caused by other factors (such as stroke, head trauma, oxidative stress, neurodegenerative disease, aging, etc.) in human life will lead to the permanent function of the neuron. Loss. However, as early as the 60s of the last century, Altman (Altman) and his colleagues questioned the reliability of this view. They reported that new neurons could continue in at least two regions of normal brain tissue in adult rats (the subgranuar zone, SGZ) and the subventricular zone (the subventricular zone, SVZ). After that, the newborn neurons in the lower hippocampus gradually migrate into the granular layer of the dentate gyrus, and the newborn neurons in the subventricular region migrate to the olfactory bulb. The discovery of alteman and Des (Das) (1965) is only more than 30 years later because of the view that there is no neuron regeneration in the brain of the born mammal. It is widely accepted that the pluripotent stem cells of mammals are located in SGZ and SVZ., which first split asymmetrically into a subprogenitor cell and a subgeneration stem cell. The former can then disintegrate into a subcellular cell that differentiates into astrocytes or neurons, and the progenitor cells maintain multiple divisions. Ability. Studies of the growth, survival and differentiation of these new cells often involve exogenous substances which are given to these markers, such as thymidine analogues or bromodeoxyglucosides (Brdu). The process of neuron regeneration in the brain of adult mammals is often strongly influenced by many internal and external factors. Endogenous external factors in the niche or stem cell niche include neural precursor cells, peripheral mature cells, intercellular interactions, cell cilia, cell secretions, and neurotransmitters. Current studies suggest that there are some specific regulation of newborn neurons in the microenvironment of SGZ and SVZ rather than in other regions of the brain. An authoritative study has shown that astrocytes in the hippocampus of adult mice promote the neurodifferentiation of adult derived hippocampal cells in vitro. As an important neurotransmitter, acetvlcholine. ACh is the earliest human neurotransmitter discovered and involved in many important functions in the organism, such as: Regulation of learning and memory, memory, mood and sleep. Although the cholinergic neurons are only distributed in some specific nuclei and regions in the brain, the axons almost dominate the whole brain tissue. For example, there is little distribution of cholinergic neurons in the hippocampus, but the cholinergic fibers from the basal forebrain are found in the hippocampus. Many studies have shown that GABA neurons, glutamic acid neurons and dopaminergic neurons play an important role in regulating the newborn neurons in the dentate gyrus of the hippocampus, but the cholinergic neurons, which are the source of the important neurotransmitters (acetylcholine), are rarely reported. [Objective] the purpose of this study mainly includes three parts: 1. the application of transgene and Specific inhibition or activation of cholinergic neurons in the basal forebrain by means of light genetic method, the effects of cholinergic neurons on the neonatal neurons in the dentate gyrus are observed. 2. the specific approach of electrophysiology, virus interference and immunohistochemistry on the above regulation,.3., presents the distribution of cholinergic neurons in the brain of mice. High definition three-dimensional images. [method] the specific inhibitory effect of cholinergic neurons on the number of new neurons in the dentate gyrus of the hippocampus: in order to specifically inhibit the cholinergic neurons in the brain of the mice, we first set up the independent Kir2.1 mouse (loxp-stop-loxp-Kir2.1-tdTomato) in the laboratory and the cholinergic neurons in the cholinergic neurons. Cre recombinant enzyme (ChAT-Cre) transgenic mice were hybridized with two kinds of transgenic mice (ChAT+/Kir2.1+, ChAT+/Kir2.1-) after multiple amplification and PCR identification. The two transgenic mice were redivided into injection tamoxifen (Tamoxifen) group and injection corn oil (Oil) group, total of four groups: ChAT+/Kir2.1+/Tamoxifen, ChAT+/Kir2.1+/Oil, ChAT. +/Kir2.1-/Tamoxifen, when ChAT+/Kir2.1-/oil. was fully expressed in Kir2.1, the mice were injected with Brdu. intraperitoneally, and then labeled at first days (d), 4D, 7d, 14d, 28d and 42d, the Brdu and counting of the hippocampal dentate gyrus (DG) were counted. Finally, the difference between each time point was counted. The specificity activated the cholinergic neurons in the basal forebrain region. To observe the recovery of the number of newborn neurons in the DG region: first, two transgenic mice (ChAT+/Kir2.1+ChR2+, ChAT+/Kir2.1+ChR2-) were obtained by hybridization of ChAT+/Kir2.1+ mice and ChR2 mice for several generations. Some male offspring were injected into the abdominal cavity with Tamoxi Fen, and then the fibers were embedded in the front of the basal brain. After the Kir2.1 and ChR2 were fully expressed, they were given. Mice were intraperitoneally injected with Brdu. from 14d to the light activation treatment of mice (the mice were divided into two groups: the mice were divided into two groups: the ChAT+/Kir2.1+ChR2+/ group and the ChAT+/Kir2.1+ChR2-/ group), and then the Brdu and counting of the DG region at 28d and 42d were marked respectively. Finally, the difference of Brdu in each group of time points was counted. The choline on the newborn neurons was observed. The expression of energy receptor: a group of 1 month old male C57BL/6 mice were selected and retrovirus (RV-GFP) was injected into the DG region. Electrophysiological tests were performed with cholinergic receptor antagonists in 4D, 7d, 14d, 21d, 28d and 42d, and the receptor type was confirmed by immunohistochemistry. The cholinergic receptor on the newborn neurons of the DG region was blocked. The effect of the number of newborn neurons in the activated model mice: Based on the above light activation model mice, the cholinergic receptors on the newborn neurons were specifically blocked by the injection retrovirus, and the Brdu of the two time points (28d and 42d) was marked and counted, and then the statistical analysis was carried out. The whole brain of the cholinergic neurons in the brain of the mice was three dimensional. Reconstruction: a 3 month old male C57BL/6 mouse was selected to slice the brain tissue in a continuous sliced sagittal plane (16 mu m). All the sagittal slices were stained by ChAT and then the brain slices were examined by a two-dimensional whole brain scan. The angle and position of all the large pictures were calibrated, and the calibrated pictures were finally carried out in three dimensions. In addition, the corresponding pictures and video are derived. In addition, the PhotoShop software is used to capture the calibrated pictures for the related areas, and then the images are synthesized and exported. [results] we found that in the study of the control of the cholinergic neurons in the DG region, we found that the cholinergic inhibition was inhibited by the cholinergic neurons. In the group, the number of Brdu in the DG region was not significantly different between the other groups at 1D, 4D, 7d and 14d, but decreased significantly at 28d and 42d. And the number of Brdu at these two time points tended to recover after giving light activation therapy. To further explore what kind of bile alkali energy receptors were involved in this regulation, we found the 4D, The newborn neurons of 7D did not respond to the stimulation of ACh, and the response to ACh stimulation at 14d, 21d and 28d showed an obvious increasing trend, and this reaction could only be blocked by the M type or M1 type acetylcholine receptor antagonist; meanwhile, our immuno histochemical results also showed that only 14d, 21d and 28d neurons could be associated with M1. The receptor was subsequently marked. Then we found that the therapeutic effect of the above-mentioned light activation was significantly weakened by blocking the M1 receptor on the newborn neurons. We successfully constructed a three-dimensional image of the distribution of cholinergic neurons in the brain of the mice. In this three-dimensional image, each nucleus of the distribution of cholinergic neurons in this three-dimensional image. The group is clearly visible, in which there are about 11000 cholinergic neurons in the the caudate putamen, about 12000 cholinergic neurons in the basal forebrain (the basal forebrain), and about 1200 cholinergic neurons in the brain stem foot bridge nucleus and the brain stem lateral tegmental nucleus (the pedunculopontine nucleus and the laterodorsal). About 2200 cholinergic neurons in the the facial nucleus of the brainstem, about 300 cholinergic neurons in the brain stem dorsal motor nucleus (the dorsal motor nucleus of the), and the number of cholinergic neurons in the whole brain (not including the cortex) are not more than 40000. [Conclusion] the cholinergic neurons in the basal forebrain region M1 receptor regulates the survival of neonatal neurons, but has no significant effect on its occurrence and division. This may provide a theoretical basis for the treatment of memory deficits in the future.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R338
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本文编号：1944922