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研究生:許珮蒨
研究生(外文):pei-chien Hsu
論文名稱:成熟與發育中大鼠三叉神經運動核與A5/A7區域正腎上腺素系統之光學與電子顯微鏡研究
論文名稱(外文):The Light and Electron Microscopic Study of Noradrenergic System in the Trigeminal Motor Nucleus and A5/A7 Area in Adult and Postnatal Rat
指導教授:閔明源楊琇雯楊琇雯引用關係
指導教授(外文):Min-Yuan MinHsiu-Wen Yang
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:140
中文關鍵詞:三叉神經運動核A5/A7 正腎上腺素系統電子顯微鏡發育
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三叉神經運動核(trigeminal motor nucleus)內含有支配控制下顎關節肌肉的運動神經元細胞本體,故其最主要之生理功能為控制節律性下顎關節運動(rhythmical jaw movements, RJMs)。三叉神經運動核之節律性下顎關節運動會受到由A5和A7正腎上腺素(noradrenergic,NAergic)神經細胞群的投射調節。本實驗之目的為探討1) 三叉神經運動核內,正腎上腺素神經纖維varicosities的超微結構, 2) 於A5/A7區域中,Naergic神經元在出生後發育上的變化,以及3) A5/A7神經元表現GABAB接受器在出生後發育上的變化。
將三叉神經運動核內之NA varicosities以免疫化學染色法標定tyrosine hydroxylase (TH,將tyrosine轉變為DOPA的酵素)。分析TH-immunoreactive(-ir) boutons的突觸連結與突觸後標的之超微結構,可觀察到有40% 的NA varicosities不會與任何神經結構形成突觸;而有60% 的NA varicosities則會與神經元之細胞本體或樹突形成對稱性或不對稱性的突觸連結。
而以免疫化學染色法分析出dopamine-b-hydroxylase(DBH,將dopamine轉變為NA的酵素)-ir之A5/A7神經元與三叉神經運動核內的NA神經纖維密度,約再P10~P20時可達到與成鼠相當的外觀形態。而利用雙重免疫螢光染色分析A5/A7神經元上GABAB接受器於出生後發育過程中的表現,結果顯示GABAB接受器的表現比率於發育過程中沒有顯著變化。
綜合實驗結果,GABA系統對A5/A7的調節於應於出生前時期即已構成。而A5/A7對三叉神經運動核的神經投射,而在出生後約P10~15左右,則可達到與成鼠相當的神經分布形態。超微結構證據顯示,NA對三叉神經運動核內之神經調控,有部份是透過 non- synaptic varicosities以擴散方式對多量標的細胞做大範圍的調節;然而,有部份synaptic varicosities則是藉由特化的突觸連結機制,對特定目標給予專一性的調控。
The trigeminal motor nucleus (Mo5) contains the cell bodies of the motoneurons innervating the jaw muscles to control the rhythmical jaw movements (RJMs). Noradrenergic (NAergic) neurons located in the pontine area, A5 and A7, project to the Mo5 and have an effect on the modulation of RJMs. The aim of the study was to investigate: 1) the ultrastructure of NAergic varicosities in the Mo5, 2) the postnatal development of the NAergic neurons in A5/A7 areas, 3) the expression of GABAB receptors on A5/A7 NAergic neurons during the postnatal ages.
NA varicosities in the Mo5 in adult rats were detected by immunohistochemistry for tyrosine hydroxylase (TH: the enzyme to convert tyrosine to DOPA). The ultrastructure of synaptic contacts, postsynaptic elements and contents formed by TH-immunoreactive (-ir) boutons were analyzed. It was found that there were 40% of NA varicosities not making synaptic contacts with any neuronal elements, and 60% of NA varicosities forming symmetrical or asymmetrical synaptic contacts with soma or dendrite.
The postnatal development of the NAergic neurons in A5/A7 areas was investigated by immunohistochemistry for dopamine-b-hydroxylase (DBH: the synthesis enzyme of NA). The adult pattern of area of A5/A7 neurons and density of NAergic fibers in Mo5 were attained by P10-20. The expression of GABAB receptors on the A5/A7 neurons was not changed at the postnatal development by immunofluorescence analysis
In conclusions, the modulation of A5/A7 neurons by GABA system was constituted at the stage before birth. The adult pattern of density of NAergic fibers raised from A5/A7 neurons were attained by P10-15. The evidence of ultrastructure suggested that noradrenaline acts on the Mo5 neurons via diffusing from non-synaptic varicosities partially to exert its influence on a large number of neurons. However, partial NAergic varicosities exert their role in modulation of specific neuronal targets via a specialized synaptic mechanism.
中文摘要……………………………………………………………… i
英文摘要……………………………………………………………….. iii
目錄…………………………………………………………………….. iv
第一章 文獻回顧與實驗目的…………………………………………. 1
第一節 三叉神經運動核系統……………………………………. 1
(一) 三叉神經運動核之位置與內在結構………………….. 1
(二) 調控三叉神經運動核傳導之神經迴路……………….. 2
第二節 A5/A7正腎上腺素系統…………………………………. 6
(一) 正腎上腺素的合成與代謝……………………………... 6
(二) 腦幹中正腎上腺素之神經投射………………………... 8
(三) A5與A7細胞本體的位置………………………………. 9
(四) A5與A7細胞的傳入神經的控制……………………... 10
(五) A5與A7細胞的傳出神經的投射……………………... 14
(六) 正腎上腺素神經纖維末梢之超微結構………………. 17
第三節 正腎上腺素對三叉神經運動核的調節………………... 21
(一) 正腎上腺素對運動機能的調節………………………. 21
(二) 正腎上腺素對三叉神經運動核的調節………………. 22
第四節 實驗目的………………………………………………... 24
第二章 材料與方法…………………………………………………... 27
第一節 動物模型與其飼養環境………………………………... 27
第二節 免疫組織化學染色法 ( Immunohistochemistry )……… 27
(一) 固定與灌流……………………………………………. 27
(二) 組織之免疫染色………………………………………. 28
(三) 資料分析………………………………………………. 28
第三節 西方墨點法(Western blotting)…………………….……. 31
(一) 樣本製備………………………………………………. 31
(二) 西方墨點法……………………………………………. 32
(三) Immunodetection ………………………………………. 32
第四節 穿透式電子顯微鏡(transmission electron microscopy)技術……………………………………………………….. 33
(一) 固定與灌流……………………………………………. 33
(二) 組織之免疫染色………………………………………. 34
(三) 電子顯微鏡標本處理…………………………………. 35
(四) 電子顯微鏡觀察………………………………………. 36
第五節 免疫螢光染色法(Immuno fluorescence)…………….…. 37
(一) 雙重免疫螢光染色……………………………………. 37
(二) 資料分析………………………………………………. 38
第六節 統計分析………………………………………………... 39
第三章 結果…………………………………………………………... 40
實驗一 成熟大白鼠中,A5/A7正腎上腺素神經核與其投射至三叉神經運動核內神經纖維的型態特徵……………….. 40
(一) A5/A7正腎上腺素細胞體之分布………………… 40
(二) A5/A7正腎上腺素神經纖維於三叉神經運動神經核中之分布…………………………………………... 41
實驗二 三叉神經運動核內正腎上腺素神經纖維末梢之超微結構……………………………………………………….. 41
(一) 以anti-TH抗體標定A7/A5正腎上腺素神經元及其神經纖維………………………………………………... 41
(二) 正腎上腺素神經纖維末梢之突觸超微結構…….…. 43
實驗三 正腎上腺素神經系統與其對三叉神經運動核投射之發 育變化………………………………………………….. 47
(一) 西方點墨法分析DBH蛋白質含量之發育變化……. 47
(二) A5/A7正腎上腺素神經元之發育變化……………… 47
(三) Mo5中正腎上腺素神經纖維密度之發育變化……... 49
(四) Mo5中正腎上腺素神經纖維varicosities之發育變化………………………………………………..……. 50
實驗四 不同出生後發育時期中,GABAB接受器在A5/A7正腎上腺素神經細胞群上的現……………………………. 51
(一) GABAB接受器在A5/A7區域中之表現的發育變化……………………………………………………... 51
(二) 不同出生發育時期之A5/A7區域中anti-GABAB與anti-DBH的雙重螢光染色………………………….. 52
第四章 討論…………………………………………………………... 55
第一節 實驗結果摘要…………………………………………... 55
第二節 三叉神經運動核內正腎上腺素神經纖維的超微結構………………………………………………….…..... 55
(一) 研究方法學上的探討…………………………….…. 56
(二) 正腎上腺素神經varicosities之突觸連結…………... 57
(三) Dense core vesicles…………………………………… 60
第三節 三叉神經運動核內正腎上腺素神經纖維的發育變化…………………………………………………..…… 61
(一) 腦幹中DBH蛋白質的發育變化……………………. 61
(二) A7/A5正腎上腺素神經元的發育變化…………….... 62
(三) 三叉神經運動核內之正腎上腺素神經纖維的發育變化………………………………………………...…… 62
第四節 A7/A5正腎上腺素神經元上表現GABAB接受器的發育變化…………………………………………………….. 64
第五節 結論與未來研究發展………...………………………… 66
(一) 結論………………………………...………………... 66
(二)未來研究發展……………………………..………….. 67
第五章 參考文獻…………………………………………………..…. 68
第六章 附圖與說明…………………………………………………... 85
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