本文選題：睡眠覺醒時相 + 腹外側視前核�。� 參考：《蘭州大學》2011年碩士論文

【摘要】：背景與目的：γ-氨基丁酸(gamma-aminobutyric acid.GABA)是中樞神經系統(tǒng)中重要的抑制性神經遞質,參與睡眠覺醒周期的轉換和慢波睡眠的維持。下丘腦腹外側視前核(ventrolateral preoptic nucleus,VLPO)內80%的神經元為GABA能,VLPO的GABA能神經元與下丘腦后部結節(jié)乳頭體核(tuberomammillary mucleus,TMN)的組織胺(histamine,HA)能神經元間投射并相互作用被認為調控睡眠覺醒的轉換。研究提示TMN也存在GABA能神經元,但是TMN的GABA能神經元在睡眠-覺醒周期中的作用目前尚不明確。本研究擬特異細胞損毀VLPO后長時間記錄大鼠睡眠覺醒周期變化,確定VLPO GABA能神經元在睡眠覺醒周期中的作用。阻止VLPO向TMN的投射后局部抑制TMN內GABA重攝,觀察對睡眠覺醒時相的影響,確定TMN內GABA能神經元在睡眠覺醒周期中的作用,分別運用組織學和免疫組織化學等方法揭示VLPO神經細胞和GABA能神經元丟失與睡眠覺醒時相變化的關系,GABA神經元與HA神經元在TMN的分布和形態(tài)特點。 方法：雄性成年SD大鼠隨機分為4組：before lesion組(n=7),after lesion組(n=7),after lesion plus vehicle組(n=8),after lesion plus GABA uptake inhibitor組(n=8)。麻醉下在額頂骨的兩側分別植入4個EEG電極,在項肌植入3根EMG電極。24小時EEG與EMG記錄和睡眠覺醒周期解析后微量注射鵝膏蕈氨酸(IA,10 nmol/0.5μl/side)于雙側VLPO行特異性細胞損毀。其中部分大鼠(after lesion組)連續(xù)記錄睡眠覺醒周期22天,余大鼠分別在損毀后第8,10,12,14天(D8,D10,D12,D14)的10：00或22：00 h注射GABA重攝抑制劑哌啶甲酸(NPA.1mmol/0.5μl/side).或生理鹽水于雙側TMN,觀察晝(08：00-20：00 h)和夜(20：00-08：00h)各12小時睡眠覺醒時相的變化,EEG快速傅里葉轉換(fast fourier transformation, FFT)行各頻段腦波power spectral density(PSD)分析.記錄后分別行Nissl染色,免疫組化標記早刻基因c-fos與谷氨酸脫羧酶(glutamate decarboxylase,GAD)觀察VLPO的細胞和GABA能神經元的損毀數(shù)量。正常大鼠(n=4)行GAD與組氨酸脫羧酶(histidine decarboxylase,HDC)免疫染色觀察TMN的HA能與GABA能神經元在分布和形態(tài)上的特點。 結果： 1.雙側損毀VLPO后對睡眠覺醒周期和皮層腦電的影響VLPO損毀后連續(xù)記錄分析睡眠覺醒周期22天,與before lesion比較,大鼠生理睡眠期(08：00-20：00 h)：覺醒(wakefulness,W)D6-D16曾加,累計平均增加39.59%(P0.01),其中D12達高峰；淺慢波睡眠(light slow wave sleep, SWS1)從D8始增加至D22,平均增加46.86%(P0.01)；深慢波睡眠(deep slow wave sleep,SWS2)D4-D22減少,平均減少50.14%(P0.01),明顯變化在第二周；異相睡眠(paradoxical sleep, PS)無統(tǒng)計學變化。皮層EEG PSDβ+γ波與a波明顯增強,δ波明顯減弱,0波變化不明顯。生理覺醒期(20：00-08：00 h)：僅第二周的w平均增加15.95%(P0.05)和SWS2減少57.5%(P0.01)。組織學和免疫組化的細胞計數(shù)結果微量注射IA導致VLPO內75-90%的GABA神經元/細胞減少,fos與GAD免疫反應陽性細胞數(shù)目與SWS2和慢波睡眠(slow wave sleep,SWS)時間呈線性相關。 2.雙側損毀VLPO后注射NPA于TMN對睡眠覺醒周期和皮層腦電的影響VLPO損毀后雙側TMN注射NPA于各時相變化明顯的D8,D10,D12和D14,與after lesion plus vehicle組相比,08:00-20:00 h:SWS1和SWS2分別減少23.53%(P0.05)和增加83.41%(P0.01),其中第12天SWS1和SWS2分別減少28.0%(P0.05)和增加158.1%(P0.01)；20：00-08：00 h：W減少20.22%(P0.05),SWS1和SWS2分別增加38.38%(P0.05)和113.1%(P0.01),其中第12天W減少22.79%(P0.05),SWS 1和SWS2分別增加52.17%(P0.05)和1165.1%(P0.01)。與before lesion組相比,08：00-20：00 h：W和SWS1分別增加25.61%(P0.05)和25.32%(P0.05),SWS2減少25.87%(P0.05);20:00-08:00 h:W減少8.90%(P0.05)和SWS1增加43.52%(P0.05),盡管有恢復趨勢,但未達到損毀前水平。皮層EEG PSDδ波與α波明顯增強,β+γ波明顯減弱,θ波變化不明顯。Waterfall Power顯示NPA注射雙側TMN45分鐘潛伏期后引發(fā)高振幅6波增強5h。 3.TMN的HA能和GABA能神經元的分布免疫熒光雙標記顯示TMN的HDC陽性神經元與GAD神經元共存,提示神經遞質HA與GABA共存于TMN神經元。 結論： IA特異細胞損毀VLPO可導致GABA能神經元丟失,導致睡眠覺醒時相的改變起始D4延續(xù)至本研究記錄D22.但變化高峰為D12,雖第2周末始有所恢復但仍未達到正常生理水平(損毀前)。VLPO損毀導致生理睡眠期(08：00-20：00h)的SWS2破碎和抑制,W和SWS1的發(fā)生次數(shù)和時間增加,而PS無變化。皮質EEGδ波power減低；生理覺醒期(20:00-08:00 h)D8-D14的W增加,SWS2減少,SWS1和PS無統(tǒng)計學變化。GABA能神經元丟失數(shù)量與SWS和SWS2的時間存在線性關系。上述結果表明VLPO的GABA能神經元與SWS的維持有關,與PS的發(fā)生和維持無關。VLPO損毀后生理睡眠期抑制TMN的GABA重攝引起SWS2增加,生理覺醒期抑制TMN的GABA重攝引起SWS1和SWS2增加,皮質EEGδ波與α波power增強,但NPA不改變VLPO損毀導致的SWS2減少,W和SWS1增加的趨勢。結果表明TMN的GABA神經元參與睡眠驅動或慢波睡眠的發(fā)生。神經遞質HA與GABA共存于TMN神經元,提示TMN的HA能神經元與GABA能神經元相互作用參與睡眠覺醒周期的調節(jié)。
[Abstract]:Background and purpose: gamma aminobutyric acid (gamma-aminobutyric acid.GABA) is an important inhibitory neurotransmitter in the central nervous system, participates in the transformation of the sleep awakening cycle and the maintenance of slow wave sleep. 80% of the neurons in the ventrolateral preoptic nucleus (VLPO) of the hypothalamus are GABA, and the GABA neurons of VLPO are and lower. The projection and interaction of tuberomammillary mucleus (TMN) in the posterior thalamic nodular nucleus (histamine, HA) can regulate the transformation of sleep awakening. The study suggests that TMN also exists in GABA ergic neurons, but the role of GABA neurons in TMN in the sleep wake cycle is not yet clear. This study is intended to be specific. After VLPO, the changes of sleep awakening cycle of rats were recorded for a long time, and the role of VLPO GABA neurons in the sleep awakening cycle was determined. After blocking VLPO to TMN, the local inhibition of GABA reactivity in TMN and the effect of the sleep awakening phase were observed, and the effect of GABA neurons in TMN in the sleep awakening cycle was determined, respectively. The relationship between the loss of VLPO neurons and GABA neurons and the change of sleep awakening, the distribution and morphological characteristics of GABA neurons and HA neurons in TMN were revealed by the methods of weave and immunohistochemistry.
Methods: male adult SD rats were randomly divided into 4 groups: before lesion group (n=7), after lesion group (n=7), after lesion plus vehicle group (n=8) and 4 electrodes implanted respectively on both sides of the frontal parietal bone. After phase analysis, microinjection of amanaminic acid (IA, 10 nmol/0.5 l/side) was damaged in bilateral VLPO specific cells. Some rats (group after lesion) recorded the sleep awakening cycle for 22 days, and the remaining rats were injected with piperidine formic formic formic acid at 10:00 or 22:00 H (D8, D10, D12, D14). L/0.5 mu l/side). Or physiological saline in bilateral TMN, observe the changes of the 12 hour sleep awakening phase of day (08:00-20:00 h) and night (20:00-08:00h), EEG fast Fourier transform (fast Fourier transformation, FFT) of each frequency band brain wave power. Gene c-fos and glutamic acid decarboxylase (glutamate decarboxylase, GAD) were used to observe the damage of VLPO cells and GABA neurons. Normal rats (n=4) were used to observe the distribution and morphology of GAD and histidine decarboxylase (histidine decarboxylase, HDC).
Result錛，

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