【作者】 高尚锋；

【导师】 包爱民；

【作者基本信息】 浙江大学 ， 神经生物学， 2012， 博士

【摘要】 第一部分：GABA能神经传递紊乱与抑郁症患者HPA轴过度激活的关系目的：下丘脑室旁核(hypothalamic paraventricular nucleus, PVN)中的促肾上腺皮质激素释放激素(corticotropin-releasing hormone, CRH)神经元的激活在情感障碍发病机制中扮演着至关重要的作用。γ-氨基丁酸(Gamma-aminobutyric acid, GABA)是主要的抑制性神经递质之一。我们的假说是抑郁症中下丘脑PVN中GABA能神经递质失调可能会导致CRH免疫反应性(immunoreactive, IR)神经元的过度激活。方法：通过免疫细胞化学和图像分析方法对14位情感障碍患者和12位对照者下丘脑PVN中的谷氨酸脱羧酶(Glutamic acid decarboxylase, GAD65/67)免疫染色进行定量分析。14位情感障碍患者中9位患有重性抑郁(major depressive disorder, MDD),5位患有双向障碍(bipolar disorder, BD)。此外,我们还对这些个体的下丘脑PVN中CRH-IR神经元总数进行了分析。结果：情感障碍患者PVN-GAD65/67呈现神经末梢点状染色分布。GAD65/67-IR结构的密度比对照组低(P=0.080),在MDD组降低更为显著(P=0.028),PVN-GAD65/67-IR表达的下降伴随着PVN-CRH-IR神经元总数的显著增加。在情感障碍组中,PVN-GAD65/67-IR结构的密度与CRH-IR神经元总数之间存在显著负相关(rho=-0.527,P=0.032,n=13),该显著相关性在对照组中不存在。结论：情感障碍患者(尤其是MDD)PVN中GABA能神经支配减弱,降低了GABA对CRH-IR神经元的抑制作用,这是抑郁症患者HPA轴活性亢进的重要机制之一。第二部分：NOS-NO系统在应激动物模型中的初步研究目的：已有研究表明,气体性神经递质一氧化氮(nitric oxide, NO)及其神经元型NO合酶(neuronal NO synthase, nNOS)在抑郁症患者大脑和血浆中发生明显改变,但是它们参与抑郁症发病的作用机制尚不清楚。本文旨在探讨NOS-NO系统在应激后大鼠下丘脑和血浆中是否改变,从而为将来研究NOS-NO系统参与抑郁症发病机制寻找恰当的动物模型和提供科学依据。方法：在成年雄性大鼠建立足底电击诱导的急性应激模型和慢性不可预知性应激(chronic unpredicted stress, CUS)模型。对照组未受到任何刺激。CUS大鼠先进行旷场实验和糖水偏好实验后取材。血浆中NO代谢物(硝酸盐和亚硝酸盐,NOx)、皮质酮(CORT)以及下丘脑NOS活性分别通过各自的商业化试剂盒进行测定。在下丘脑室旁核(paraventricular nucleus, PVN)中进行CRH和nNOS免疫细胞化学研究,应用图像分析软件进行定量分析。nNOS与CRH、血管加压素、催产素等神经肽在PVN的共定位关系通过免疫荧光双标实验观察。结果：血浆中CORT水平在足底电击0、5、15和30min后显著升高(P≤0.038),血浆中NOx水平分别在0和30min出现高峰(P≤0.005)。下丘脑PVN中CRH-IR于足底电击后15min(P=0.024)和30min(P=0.022)明显增强,而nNOS-IR于足底电击后15min明显减弱(P=0.030)。下丘脑总NOS活性在足底电击后15min明显下降(P=0.028)。CUS大鼠在旷场和糖水偏好实验中表现出抑郁焦虑样行为,血浆中CORT(P=0.001)和NOx(P=0.001)水平均显著升高,下丘脑总NOS活性在CUS后明显下降(P=0.025),下丘脑PVN中nNOS阳性细胞密度在CUS组中显著下降(P=0.019),并且该显著性降低在nNOS强阳性细胞和nNOS弱阳性细胞上均有所体现。nNOS-IR主要与催产素神经元共存,而几乎不表达于CRH-IR神经元。结论：在足底电击诱导的急性应激反应和CUS诱导的抑郁症动物模型中,下丘脑总NOS的活性和PVN-nNOS表达显著降低,血浆NO水平却明显升高。此外,足底电击后nNOS-IR并不与CRH-IR神经元共存,而是主要表达在催产素细胞上。因此,nNOS与催产素神经元的相互作用将是研究nNOS参与应激反应机制的重要线索之一。第三部分：NOS-NO系统通过改变GABA能神经传递参与抑郁症患者前额叶皮层功能调节目的：前额叶皮层(prefrontal cortex, PFC)在结构和功能上的异常与抑郁症的病因学和症状学密切相关。近年来研究表明,气体性神经递质一氧化氮(nitric oxide, NO)在抑郁症病理过程中发挥重要作用,但是它是否参与并如何调节PFC的活性目前还不清楚。因此,本文旨在研究抑郁症患者脑脊液(cerebrospinal fluid, CSF)中NO含量和PFC中NO合酶(NO synthase),即神经型NOS (neuronal NOS)、内皮型NOS (endothelial NOS)和诱导型NOS (iNOS)的表达。此外,我们还研究了选择性阻断nNOS来源的NO产生对PFC-GABA能神经元活性的影响。方法：通过测定CSF内NO代谢产物,即硝酸盐和亚硝酸盐(NO、)的含量来反映抑郁症患者和匹配的对照者CSF中NO水平。采用实时定量PCR技术检测背外侧PFC (dorsolateral PFC, DLPFC)和前扣带回皮层(anterior cingulate cortex, ACC)中的nNOS.eNOS和iNOS的mRNA水平。应用免疫细胞化学方法和图像分析软件在ACC中定量分析nNOS蛋白水平,并通过免疫荧光双标技术观察nNOS与谷氨酸脱羧酶(GAD65/67)的共定位情况。此外,应用nNOS的选择性抑制剂7-nitroindazole(7一NI)处理小鼠ACC脑片,观察其对GABA能神经元电生理活动的改变。结果：抑郁症组CSF-NOx水平显著低于对照组(P=0.007)。抑郁症患者ACC中,而不是DLPFC中,nNOS-mRNA水平具有降低的趋势(P=0.083).nNOS-IR王要分布在正常人脑ACC的Ⅱ/Ⅲ层。在抑郁症患者组中,ACC-nNOS-IR在整个灰质中的细胞密度和平均光密度均降低(P=0.083),尤其是ACC的Ⅱ/Ⅲ层降低更为显著(P=0.043)。对照组nNOS-IR在ACC灰质中的细胞密度与CSF-NOx水平呈显著正相关(rho=0.667,P=0.050,n=9).nNOS与GABA能神经元的标记物GAD65/67在抑郁症患者ACC中存在明显共定位。此外,nNOS选择性抑制剂7-NI与ACC脑片孵育显著增加了GABA介导的抑制性突触后电流(mIPSCs)的频率(P<0.05, n=11),但未改变mIPSCs的幅度。结论：抑郁症患者ACC-nNOS表达下调,CSF-NOx水平减低,选择性抑制小鼠ACC中的nNOS活性显著影响了GABA能神经传递。更多还原

【Abstract】 Part Ⅰ:Dysregulation of GABAergic neurotransmission and the hyperactivated HPA axis in depressionObjectives:Activation of corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) is crucial in the pathogenesis of mood disorder. Gamma-aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters. We hypothesized that dysregulation of GABAergic neurotransmission contributes to the hyperactivity of CRH-immunoreactive (IR) neurons in depression.Methods:Glutamic acid decarboxylase (GAD)65/67-IR boutons were quantified in the postmortem PVN by image analysis in14mood disorder patients (9major depressive disorder, MDD,5bipolar disorder, BD) and12controls. For some of these subjects the total number of CRH-IR neurons in the PVN was also analyzed.Results:The density of GAD65/67-positive structures decreased in mood disorder patients (P=0.080), especially in MDD group (P=0.028), compared to the matched controls. The reduction in GAD65/67-IR was accompanied by significantly increased numbers of CRH-IR neurons. In addition, a negative correlation between PVN-GAD65/67-IR density and CRH-IR neuron numbers was found in the mood disorder group (rho=-0.527, P=0.032, n=13), but not in the control group.Conclusions:A diminished GABAergic input to the PVN may contribute to the activation of CRH-IR neurons in depression, most prominently in MDD. This is an important factor that leads to the overeactivity of HPA axis in depression. Part Ⅱ:A preliminary study on the alteration of NOS-NO system in stress animal modelsObjective: The gaseous neurotransmitter nitric oxide (NO) and neuronal NO synthase (nNOS) were reported to be altered in the brain and plasma in depression, but the underlying mechanism remains unknown. Therefore, we investigated in stress models the changes of NOS-NO pathway in the rat hypothalamus and plasma so as to search for an ideal model and provide scientific evidence for future research.Methods:Adult male rats were used to establish chronic unpredicted stress (CUS) model and acute stress model induced by foot shock, none of any stimuli was subjected to control rats. Open field test and sucrose preference test were performed on CUS rats. Plasma NO metabolites, plasma corticosterone (CORT) levels, and hypothalamic NOS activity were determined by respective commercial kits. The density of CRH-and nNOS-expressing cells was quantified in the hypothalamic paraventricular nucleus (PVN) by image analysis. Co-localization between nNOS and the neuropeptides, i.e. CRH, vasopressin and oxytocin in the PVN was observed by double immunofluorescence.Results:Significant increases of plasma CORT were observed after foot shock at0,5,15and30min (P<0.038), and for levels of NO metabolites at0and30min (P<0.005). The density of CRH-immunoreactivity (IR) increased at15min (P=0.024) and30min (P=0.022), while nNOS-IR deceased at15min (P=0.030) in the hypothalamic PVN after foot shock. The total NOS activity decreased at15min in the hypothalamus after foot shock (P=0.028). CUS rats showed depression-and anxiety-like behaviors in open field test and sucrose preference test. CUS rats had higher CORT (P=0.001) and NOX (P=0.001) levels in plasma than control rats. In the CUS model, nNOS-IR cell density significantly decreased in the hypothalamic PVN (P=0.019), and this reduction was based upon both strongly and weakly stained neurons. The total NOS activity significantly decreased in the hypothalamus of CUS rats (P=0.025). nNOS-IR was mainly expressed in oxytocin-positive neurons, but hardly colocalized with CRH-IR neurons.Conclusion:The present study showed a decrease of cNOS activity and PVN-nNOS expression in the hypothalamus, but an increase of NO levels in plasma both in foot shock-induced acute stress and CUS-caused depression models. In addition, nNOS did not express in CRH-IR neurons, but in oxytocin-IR neurons after foot shock. The interaction between nNOS and oxytocin will thus be a promising clue to reveal the role of nNOS in stress responses. Part Ⅲ:NOS-NO system is involved in the regulation of prefrontal cortex function by affecting GABAergic neurotransmission in depression Objective: Alterations in prefrontal cortex (PFC) are crucially involved in the etiology and symptoms of depression. A key role of the gaseous neurotransmitter nitric oxide (NO) has also been proposed, but whether and how NO affect the PFC activity are largely unknown in depression. The present study, therefore, aimed to determine the putative changes of brain NO production, and the transcriptional and/or protein level of the isoforms of the NO synthase (NOS), i.e. neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) in the PFC in depression. Meanwhile, we investigated the effects of NO on GABAergic neuronal activity by selectively blocking the nNOS activity.Methods:The levels of NO, as determined by its metabolites nitrate and nitrite (NOx), were measured in the postmortem ventricular cerebrospinal fluid (CSF) of depressed patients and matched controls. The mRNA levels of nNOS, eNOS and iNOS were measured by real-time PCR in the dorsolateral PFC (DLPFC) and in the anterior cingulate cortex (ACC) of depressed patients. ACC-nNOS immunoreactivity (IR) was assessed by immunocytochemistry and image analysis, and the colocalization between nNOS and GAD65/67was observed by double immunofluorescence. Moreover,7-nitroindazole, the selective nNOS inhibitor, was applied to ACC slices and observed the effects of nNOS-derived NO on the electrophysiological activity GABAergic neurons.Results:We found a significant decrease of CSF-NOx levels in the depressed patients compared to controls (P=0.007). There was a trend toward a lower nNOS mRNA levels in depression in ACC (P=0.083), but not in DLPFC (P=0.939). nNOS-IR was observed to be mainly distributed in the layer Ⅱ/Ⅲ of the human ACC, and a remarkable decrease of nNOS-IR in the grey matter of ACC (P=0.083), especially in layer II/III (P=0.043), was found in depressed patients. A significant positive correlation between the CSF-NOx levels and the ACC-nNOS-IR cell numbers was found in the control group (rho=0.667, P=0.050, n=9). Co-localization between nNOS and GAD65/67, the marker of GABAergic neurons, was found in the human ACC. Furthermore, bath application of7-NI in ACC slices significantly enhanced the frequency of GABAergic mIPSCs (P<0.05; n=11), but did not significantly alter mIPSC amplitude.Conclusion:A diminished ACC-nNOS expression and a decreased CSF-NOX level were found in depression. Selectively inhibiting nNOS activity had a significant effect on GABAergic neurotransmission in mouse ACC slices.更多还原