本文選題：膽堿能神經元 + 新生神經元　； 參考：《華中科技大學》2016年博士論文

【摘要】：[背景]上世紀90年代以前,研究人員普遍認為在成年哺乳動物腦中的神經元不能再生。因此,人們認為在人的一生中,任何由于其它因素(如：中風、頭部創(chuàng)傷、氧化應激、神經退行性疾病、衰老等)導致的神經元死亡,都將導致其功能的永久喪失。然而,早在上世紀60年代,阿爾特曼(Altman)和其同事就質疑了這一觀點的可靠性。他們報道,在成年大鼠正常腦組織的至少兩個區(qū)域(海馬顆粒下層(the subgranuar zone,SGZ)和側腦室下區(qū)(the subventricular zone, SVZ))內,新生神經元能持續(xù)不斷地產生。之后,海馬顆粒下層的新生神經元逐漸遷移到齒狀回的顆粒層內,側腦室下區(qū)的新生神經元則遷移到嗅球。由于受出生后的哺乳動物大腦中不存在神經元再生這種觀點的影響,使得阿爾特曼和戴斯(Das) (1965)的發(fā)現(xiàn)三十多年后才被大家廣泛接受。哺乳動物的多能神經干細胞位于SGZ和SVZ。這些干細胞首先不對稱地分裂為一個子代祖細胞和一個子代干細胞。然后前者能再不對稱地分裂為可分化成星形膠質細胞或神經元的子代細胞,并且祖細胞保持有多次分裂的能力。對這些新生細胞生長、存活和分化的研究,常常涉及到外源性給予胸苷類似物或溴脫氧尿甙(Brdu)這些可標記分裂細胞的物質。成年哺乳動物大腦中的神經元再生過程常受到許多內在和外在因素的強烈影響。局部微環(huán)境(“神經源性壁龕”或“干細胞壁龕”)中的內源性外在因素包括：神經前體細胞、周邊成熟細胞、細胞間相互作用、細胞纖毛、細胞分泌物和神經遞質�，F(xiàn)在的研究認為在SGZ和SVZ的微環(huán)境中,而不是大腦其它區(qū)域,存在一些特定的調控新生神經元生長發(fā)育的因素。一項權威的研究表明在成年小鼠海馬中的星形膠質細胞促進體外成年源性海馬祖細胞的神經分化。乙酰膽堿(acetvlcholine. ACh)作為一種重要的神經遞質,它是被人類最早發(fā)現(xiàn),并在生物體內參與很多重要功能的遞質,如：調控學習與記憶、情緒和睡眠等。雖然膽堿能神經元胞體只分布在大腦中一些特定的核團和區(qū)域,但其軸突幾乎支配整個大腦組織。例如,海馬中雖然幾乎沒有膽堿能神經元胞體的分布,但其間卻遍布來自基底前腦的膽堿能神經纖維。許多研究表明GABA能神經元、谷氨酸能神經元和多巴胺能神經元對海馬齒狀回區(qū)的新生神經元具有重要的調控作用,但作為重要神經遞質(乙酰膽堿)來源的膽堿能神經元卻鮮有報道。[目的]本研究的目的主要包括三部分：1.應用轉基因和光遺傳手段特異性抑制或激活基底前腦膽堿能神經元,觀察膽堿能神經元對海馬齒狀回區(qū)新生神經元的調控作用2.運用電生理、病毒干擾和免疫組織化學等技術探討上述調控作用的具體途徑。3.完美呈現(xiàn)小鼠大腦中膽堿能神經元分布的高清三維圖像。[方法]特異性抑制膽堿能神經元對海馬齒狀回區(qū)新生神經元數(shù)目的調控作用：為了特異性抑制小鼠大腦內的膽堿能神經元,我們首先將實驗室自主構建的Kir2.1工具鼠(loxp-stop-loxp-Kir2.1-tdTomato)與在膽堿能神經元中表達Cre重組酶的(ChAT-Cre)轉基因小鼠雜交,經多次擴增雜交和PCR鑒定后得到兩種轉基因小鼠(ChAT+/Kir2.1+,ChAT+/Kir2.1-)。將這兩種轉基因小鼠再分為注射他莫昔芬(Tamoxifen)組和注射玉米油(Oil)組,共計四組：ChAT+/Kir2.1+/Tamoxifen,ChAT+/Kir2.1+/Oil,ChAT+/Kir2.1-/Tamoxifen, ChAT+/Kir2.1-/oil。待Kir2.1充分表達后,給各組小鼠腹腔注射Brdu.然后分別標記在第1天(d)、4d、7d、14d、28d和42d時海馬齒狀回區(qū)(DG)的Brdu并計數(shù)。最后統(tǒng)計在各個時間點各組間Brdu數(shù)量的差異。特異性激活基底前腦區(qū)膽堿能神經元并觀察DG區(qū)新生神經元數(shù)目的恢復情況：首先將ChAT+/Kir2.1+小鼠和ChR2小鼠雜交數(shù)代,得到兩種轉基因小鼠(ChAT+/Kir2.1+ChR2+,ChAT+/Kir2.1+ChR2-)。選取一些雄性后代進行Tamoxi fen腹腔注射,然后在其基底前腦部埋置光纖。待Kir2.1和ChR2充分表達后,給各組小鼠腹腔注射Brdu。從14d開始對小鼠給予光激活處理(實驗小鼠分為兩組：ChAT+/Kir2.1+ChR2+/給光組、ChAT+/Kir2.1+ChR2-/給光組),然后分別標記28d和42d時DG區(qū)的Brdu并計數(shù)。最后統(tǒng)計在各個時間點各組間Brdu數(shù)量的差異。觀察新生神經元上膽堿能受體的表達情況：選取一批1月齡的雄性C57BL/6小鼠,在其DG區(qū)注射逆轉錄病毒(RV-GFP)。分別于4d、7d、14d、21d、28d和42d時結合膽堿能受體拮抗劑進行電生理檢測,以及對受體類型進行免疫組化標記確證。阻斷DG區(qū)新生神經元上膽堿能受體對光激活模型小鼠中新生神經元數(shù)目的影響：基于上述光激活模型小鼠,通過注射逆轉錄病毒特異性阻斷新生神經元上膽堿能受體,標記上述兩個時間點(28d和42d)的Brdu并計數(shù),然后進行統(tǒng)計分析。小鼠大腦中膽堿能神經元分布的全腦三維重構：選取一只3月齡的雄性C57BL/6小鼠,對其腦組織進行矢狀面的連續(xù)切片(16μm)。對所有矢狀面腦片進行ChAT免疫組化染色,然后對腦片進行二維的全腦掃圖。對所有拍攝好的大圖進行角度和位置的校準,最后將校準好的圖片進行三維合成,并導出相應的圖片和視頻。此外,針對相關區(qū)域,首先用PhotoShop軟件在校準好的圖片上進行截圖,然后將這些裁剪出的圖片進行三維合成并導出數(shù)據(jù)。[結果]在觀察膽堿能神經元對DG區(qū)新生神經元調控的研究中,我們發(fā)現(xiàn)在膽堿能抑制組中,DG區(qū)Brdu的數(shù)目在1d、4d、7d和14d時和其它組間沒有明顯差異,而在28d和42d時則顯著降低。并且在給予光激活治療后,這兩個時間點的Brdu數(shù)目則趨向復原。為了進一步探討是哪種(或哪類)膽堿能受體參與了這種調控作用,我們發(fā)現(xiàn)第4d、7d的新生神經元對ACh的刺激均沒有反應,而在14d、21d和28d時對ACh的刺激有反應,且呈明顯的遞增趨勢,并且這種反應只能被M型或M1型乙酰膽堿受體拮抗劑阻斷;同時,我們的免疫組織化學結果也顯示,只有14d、21d和28d時的新生神經元能與M1受體共標。隨后我們通過特異性阻斷新生神經元上的M1型受體,發(fā)現(xiàn)上述光激活的治療作用明顯減弱。我們首次應用免疫組織化學技術成功構建了小鼠大腦中膽堿能神經元分布的三維圖像。在此三維圖像中,膽堿能神經元分布的各個核團清晰可見,其中：尾殼核(the caudate putamen)中有約11000個膽堿能神經元、基底前腦(the basal forebrain)中約12000個膽堿能神經元、腦干腳橋核與腦干側被蓋核(the pedunculopontine nucleus and the laterodorsal tegmental nucleus)中約1200個膽堿能神經元、腦干面神經核(the facial nucleus of the brainstem)中約2200個膽堿能神經元、腦干背運動核(the dorsal motor nucleus of the brainstem)中約300個膽堿能神經元,并且全腦中(不包含皮質)膽堿能神經元的數(shù)目不超過40000個。[結論]基底前腦區(qū)的膽堿能神經元通過M1受體調控新生神經元的存活,而對其發(fā)生和分裂則沒有明顯影響,這可能為今后治療記憶缺陷提供理論依據(jù)。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R338
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本文編號：1944922