损毁腹外侧视前核后抑制结节乳头体核γ-氨基丁酸重摄对大鼠睡眠—觉醒时相的影响

发布时间：2018-04-24 00:35

  本文选题：睡眠觉醒时相 + 腹外侧视前核　； 参考：《兰州大学》2011年硕士论文

【摘要】：背景与目的：γ-氨基丁酸(gamma-aminobutyric acid.GABA)是中枢神经系统中重要的抑制性神经递质,参与睡眠觉醒周期的转换和慢波睡眠的维持。下丘脑腹外侧视前核(ventrolateral preoptic nucleus,VLPO)内80%的神经元为GABA能,VLPO的GABA能神经元与下丘脑后部结节乳头体核(tuberomammillary mucleus,TMN)的组织胺(histamine,HA)能神经元间投射并相互作用被认为调控睡眠觉醒的转换。研究提示TMN也存在GABA能神经元,但是TMN的GABA能神经元在睡眠-觉醒周期中的作用目前尚不明确。本研究拟特异细胞损毁VLPO后长时间记录大鼠睡眠觉醒周期变化,确定VLPO GABA能神经元在睡眠觉醒周期中的作用。阻止VLPO向TMN的投射后局部抑制TMN内GABA重摄,观察对睡眠觉醒时相的影响,确定TMN内GABA能神经元在睡眠觉醒周期中的作用,分别运用组织学和免疫组织化学等方法揭示VLPO神经细胞和GABA能神经元丢失与睡眠觉醒时相变化的关系,GABA神经元与HA神经元在TMN的分布和形态特点。 方法：雄性成年SD大鼠随机分为4组：before lesion组(n=7),after lesion组(n=7),after lesion plus vehicle组(n=8),after lesion plus GABA uptake inhibitor组(n=8)。麻醉下在额顶骨的两侧分别植入4个EEG电极,在项肌植入3根EMG电极。24小时EEG与EMG记录和睡眠觉醒周期解析后微量注射鹅膏蕈氨酸(IA,10 nmol/0.5μl/side)于双侧VLPO行特异性细胞损毁。其中部分大鼠(after lesion组)连续记录睡眠觉醒周期22天,余大鼠分别在损毁后第8,10,12,14天(D8,D10,D12,D14)的10：00或22：00 h注射GABA重摄抑制剂哌啶甲酸(NPA.1mmol/0.5μl/side).或生理盐水于双侧TMN,观察昼(08：00-20：00 h)和夜(20：00-08：00h)各12小时睡眠觉醒时相的变化,EEG快速傅里叶转换(fast fourier transformation, FFT)行各频段脑波power spectral density(PSD)分析.记录后分别行Nissl染色,免疫组化标记早刻基因c-fos与谷氨酸脱羧酶(glutamate decarboxylase,GAD)观察VLPO的细胞和GABA能神经元的损毁数量。正常大鼠(n=4)行GAD与组氨酸脱羧酶(histidine decarboxylase,HDC)免疫染色观察TMN的HA能与GABA能神经元在分布和形态上的特点。 结果： 1.双侧损毁VLPO后对睡眠觉醒周期和皮层脑电的影响VLPO损毁后连续记录分析睡眠觉醒周期22天,与before lesion比较,大鼠生理睡眠期(08：00-20：00 h)：觉醒(wakefulness,W)D6-D16曾加,累计平均增加39.59%(P0.01),其中D12达高峰；浅慢波睡眠(light slow wave sleep, SWS1)从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减少,平均减少50.14%(P0.01),明显变化在第二周；异相睡眠(paradoxical sleep, PS)无统计学变化。皮层EEG PSDβ+γ波与a波明显增强,δ波明显减弱,0波变化不明显。生理觉醒期(20：00-08：00 h)：仅第二周的w平均增加15.95%(P0.05)和SWS2减少57.5%(P0.01)。组织学和免疫组化的细胞计数结果微量注射IA导致VLPO内75-90%的GABA神经元/细胞减少,fos与GAD免疫反应阳性细胞数目与SWS2和慢波睡眠(slow wave sleep,SWS)时间呈线性相关。 2.双侧损毁VLPO后注射NPA于TMN对睡眠觉醒周期和皮层脑电的影响VLPO损毁后双侧TMN注射NPA于各时相变化明显的D8,D10,D12和D14,与after lesion plus vehicle组相比,08:00-20:00 h:SWS1和SWS2分别减少23.53%(P0.05)和增加83.41%(P0.01),其中第12天SWS1和SWS2分别减少28.0%(P0.05)和增加158.1%(P0.01)；20：00-08：00 h：W减少20.22%(P0.05),SWS1和SWS2分别增加38.38%(P0.05)和113.1%(P0.01),其中第12天W减少22.79%(P0.05),SWS 1和SWS2分别增加52.17%(P0.05)和1165.1%(P0.01)。与before lesion组相比,08：00-20：00 h：W和SWS1分别增加25.61%(P0.05)和25.32%(P0.05),SWS2减少25.87%(P0.05);20:00-08:00 h:W减少8.90%(P0.05)和SWS1增加43.52%(P0.05),尽管有恢复趋势,但未达到损毁前水平。皮层EEG PSDδ波与α波明显增强,β+γ波明显减弱,θ波变化不明显。Waterfall Power显示NPA注射双侧TMN45分钟潜伏期后引发高振幅6波增强5h。 3.TMN的HA能和GABA能神经元的分布免疫荧光双标记显示TMN的HDC阳性神经元与GAD神经元共存,提示神经递质HA与GABA共存于TMN神经元。 结论： IA特异细胞损毁VLPO可导致GABA能神经元丢失,导致睡眠觉醒时相的改变起始D4延续至本研究记录D22.但变化高峰为D12,虽第2周末始有所恢复但仍未达到正常生理水平(损毁前)。VLPO损毁导致生理睡眠期(08：00-20：00h)的SWS2破碎和抑制,W和SWS1的发生次数和时间增加,而PS无变化。皮质EEGδ波power减低；生理觉醒期(20:00-08:00 h)D8-D14的W增加,SWS2减少,SWS1和PS无统计学变化。GABA能神经元丢失数量与SWS和SWS2的时间存在线性关系。上述结果表明VLPO的GABA能神经元与SWS的维持有关,与PS的发生和维持无关。VLPO损毁后生理睡眠期抑制TMN的GABA重摄引起SWS2增加,生理觉醒期抑制TMN的GABA重摄引起SWS1和SWS2增加,皮质EEGδ波与α波power增强,但NPA不改变VLPO损毁导致的SWS2减少,W和SWS1增加的趋势。结果表明TMN的GABA神经元参与睡眠驱动或慢波睡眠的发生。神经递质HA与GABA共存于TMN神经元,提示TMN的HA能神经元与GABA能神经元相互作用参与睡眠觉醒周期的调节。
[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).
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