神经递质GABA和NO在抑郁症患者和应激模型中的病理意义
发布时间：2018-09-02 08:42
【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(P0.05, 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。
[Abstract]:Part one: the relationship between GABA nerve transmission disturbance and HPA axis overactivation in depressive patients
AIM: Activation of corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) plays an important role in the pathogenesis of affective disorders. Gamma-aminobutyric acid (GABA) is the main inhibitory agent. Our hypothesis is that the imbalance of GABAergic neurotransmitters in hypothalamic PVN in depression may lead to excessive activation of CRH immunoreactive (IR) neurons. METHODS: Glutamate decarboxylase (G-decarboxylase) in hypothalamic PVN was detected by immunocytochemistry and image analysis in 14 patients with affective disorders and 12 controls. Immunostaining with lutamic acid decarboxylase (GAD65/67) was used to quantitatively analyze the number of CRH-IR neurons in PVN of hypothalamus in 9 of 14 patients with affective disorder (MDD) and 5 with bipolar disorder (BD).
Results: The distribution of PVN-GAD65/67 in patients with affective disorders was dot-like. The density of PVN-GAD65/67-IR structure was lower than that of the control group (P=0.080), and the decrease of PVN-GAD65/67-IR expression was more significant in the MDD group (P=0.028). The decrease of PVN-GAD65/67-IR expression was accompanied by a significant increase of the total number of PVN-CRH-IR neurons. There was a significant negative correlation between the total number of CRH-IR neurons (rho=-0.527, P=0.032, n=13), which did not exist in the control group.
Conclusion: The decrease of GABAergic innervation in PVN of patients with affective disorders (especially MDD) can decrease the inhibitory effect of GABA on CRH-IR neurons, which is one of the important mechanisms of HPA axis hyperactivity in depressive patients.
The second part: a preliminary study of NOS-NO system in stress animal models.
AIM: Previous studies have shown that nitric oxide (NO) and its neuronal nitric oxide synthase (nNOS) are significantly altered in the brain and plasma of depressive patients, but the mechanism of their involvement in the pathogenesis of depression is still unclear. Whether the hypothalamus and plasma are altered provides a scientific basis for the future study of NOS-NO system involved in the pathogenesis of depression.
METHODS: Acute and chronic unpredictable stress (CUS) models induced by plantar electric shock were established in adult male rats. The control group was not stimulated by any stimulation. After open-field experiment and sugar preference experiment, the plasma NO metabolites (nitrate and nitrite, NO), corticosterone (C The activity of NOS in hypothalamus and ORT was measured by commercial kits. CRH and nNOS immunocytochemistry were studied in paraventricular nucleus (PVN) of hypothalamus, and quantitative analysis was performed by image analysis software. Experimental observation of double fluorescence of disease.
Results: The plasma CORT level increased significantly (P < 0.038) at 0, 5, 15 and 30 minutes after plantar shock, and the plasma NOX level peaked at 0 and 30 minutes respectively (P < 0.005). CRH-IR in hypothalamic PVN increased significantly at 15 minutes (P = 0.024) and 30 minutes (P = 0.022) after plantar shock, while nNOS-IR decreased significantly at 15 minutes (P = 0.030). The activity of total NOS in hypothalamus decreased significantly after CUS (P = 0.025) and the density of nNOS positive cells in PVN in hypothalamus decreased significantly (P = 0.025) in CUS group. NNOS-IR mainly coexisted with oxytocin neurons, but hardly expressed in CRH-IR neurons.
CONCLUSION: Total NOS activity and PVN-nNOS expression in hypothalamus were significantly decreased and plasma NO level was significantly elevated in rats with acute stress induced by plantar electric shock and depression induced by CUS. In addition, nNOS-IR did not coexist with CRH-IR neurons, but mainly expressed in oxytocin cells after plantar electric shock. Neuronal interaction is one of the important clues to study the mechanism of nNOS involved in stress response.
Part III: NOS-NO system participates in the regulation of prefrontal cortex function in depressive patients by altering GABAergic neurotransmission
Objective: The structural and functional abnormalities of prefrontal cortex (PFC) are closely related to the etiology and symptoms of depression. Recent studies have shown that nitric oxide (NO) plays an important role in the pathological process of depression, but whether it participates in and how to regulate the activity of PFC. Therefore, the aim of this study was to investigate the content of NO in cerebrospinal fluid (CSF) and the expression of NO synthase (NOS), endothelial NOS and inducible NOS (iNOS) in PFC-GABA in depressed patients. Methods: The levels of NO in CSF of depressive patients and matched controls were measured by measuring the contents of nitrate and nitrite metabolites in CSF. Real-time quantitative PCR was used to detect dorsolateral PFC (DLPFC) and anterior cingulate cortex (ACC). The levels of nNOS, eNOS and iNOS mRNA were measured by immunocytochemistry and image analysis software in ACC. The co-localization of nNOS and glutamate decarboxylase (GAD65/67) was observed by immunofluorescence double labeling technique. In addition, the selective inhibitor of nNOS, 7-nitroindazole (7-NI), was used to treat ACC slices of mice. The changes in electrophysiological activity of GABA neurons were observed.
Results: The level of CSF-NOx in depression group was significantly lower than that in control group (P = 0.007). In ACC, but not DLPFC, the level of nNOS-mRNA was decreased (P = 0.083). The nNOS-IR kingdom was distributed in layer II/III of ACC in normal human brain. In depression group, the cell density and average optical density of ACC-nNOS-IR in the whole gray matter were decreased (P = 0.083). In control group, the cell density of nNOS-IR in ACC gray matter was positively correlated with CSF-NOx level (rho = 0.667, P = 0.050, n = 9). There was significant co-localization between nNOS and GABAergic neuron marker GAD65/67 in ACC of depressive patients. Slice incubation significantly increased the frequency of GABA-mediated inhibitory postsynaptic currents (mIPSCs) (P 0.05, n=11), but did not change the amplitude of mIPSCs.
CONCLUSION: The expression of ACC-nNOS is down-regulated and the level of CSF-NOx is down-regulated in depressed patients. Selective inhibition of nNOS activity in ACC of mice significantly affects GABAergic neurotransmission.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.4
本文编号：2218840
[Abstract]:Part one: the relationship between GABA nerve transmission disturbance and HPA axis overactivation in depressive patients
AIM: Activation of corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) plays an important role in the pathogenesis of affective disorders. Gamma-aminobutyric acid (GABA) is the main inhibitory agent. Our hypothesis is that the imbalance of GABAergic neurotransmitters in hypothalamic PVN in depression may lead to excessive activation of CRH immunoreactive (IR) neurons. METHODS: Glutamate decarboxylase (G-decarboxylase) in hypothalamic PVN was detected by immunocytochemistry and image analysis in 14 patients with affective disorders and 12 controls. Immunostaining with lutamic acid decarboxylase (GAD65/67) was used to quantitatively analyze the number of CRH-IR neurons in PVN of hypothalamus in 9 of 14 patients with affective disorder (MDD) and 5 with bipolar disorder (BD).
Results: The distribution of PVN-GAD65/67 in patients with affective disorders was dot-like. The density of PVN-GAD65/67-IR structure was lower than that of the control group (P=0.080), and the decrease of PVN-GAD65/67-IR expression was more significant in the MDD group (P=0.028). The decrease of PVN-GAD65/67-IR expression was accompanied by a significant increase of the total number of PVN-CRH-IR neurons. There was a significant negative correlation between the total number of CRH-IR neurons (rho=-0.527, P=0.032, n=13), which did not exist in the control group.
Conclusion: The decrease of GABAergic innervation in PVN of patients with affective disorders (especially MDD) can decrease the inhibitory effect of GABA on CRH-IR neurons, which is one of the important mechanisms of HPA axis hyperactivity in depressive patients.
The second part: a preliminary study of NOS-NO system in stress animal models.
AIM: Previous studies have shown that nitric oxide (NO) and its neuronal nitric oxide synthase (nNOS) are significantly altered in the brain and plasma of depressive patients, but the mechanism of their involvement in the pathogenesis of depression is still unclear. Whether the hypothalamus and plasma are altered provides a scientific basis for the future study of NOS-NO system involved in the pathogenesis of depression.
METHODS: Acute and chronic unpredictable stress (CUS) models induced by plantar electric shock were established in adult male rats. The control group was not stimulated by any stimulation. After open-field experiment and sugar preference experiment, the plasma NO metabolites (nitrate and nitrite, NO), corticosterone (C The activity of NOS in hypothalamus and ORT was measured by commercial kits. CRH and nNOS immunocytochemistry were studied in paraventricular nucleus (PVN) of hypothalamus, and quantitative analysis was performed by image analysis software. Experimental observation of double fluorescence of disease.
Results: The plasma CORT level increased significantly (P < 0.038) at 0, 5, 15 and 30 minutes after plantar shock, and the plasma NOX level peaked at 0 and 30 minutes respectively (P < 0.005). CRH-IR in hypothalamic PVN increased significantly at 15 minutes (P = 0.024) and 30 minutes (P = 0.022) after plantar shock, while nNOS-IR decreased significantly at 15 minutes (P = 0.030). The activity of total NOS in hypothalamus decreased significantly after CUS (P = 0.025) and the density of nNOS positive cells in PVN in hypothalamus decreased significantly (P = 0.025) in CUS group. NNOS-IR mainly coexisted with oxytocin neurons, but hardly expressed in CRH-IR neurons.
CONCLUSION: Total NOS activity and PVN-nNOS expression in hypothalamus were significantly decreased and plasma NO level was significantly elevated in rats with acute stress induced by plantar electric shock and depression induced by CUS. In addition, nNOS-IR did not coexist with CRH-IR neurons, but mainly expressed in oxytocin cells after plantar electric shock. Neuronal interaction is one of the important clues to study the mechanism of nNOS involved in stress response.
Part III: NOS-NO system participates in the regulation of prefrontal cortex function in depressive patients by altering GABAergic neurotransmission
Objective: The structural and functional abnormalities of prefrontal cortex (PFC) are closely related to the etiology and symptoms of depression. Recent studies have shown that nitric oxide (NO) plays an important role in the pathological process of depression, but whether it participates in and how to regulate the activity of PFC. Therefore, the aim of this study was to investigate the content of NO in cerebrospinal fluid (CSF) and the expression of NO synthase (NOS), endothelial NOS and inducible NOS (iNOS) in PFC-GABA in depressed patients. Methods: The levels of NO in CSF of depressive patients and matched controls were measured by measuring the contents of nitrate and nitrite metabolites in CSF. Real-time quantitative PCR was used to detect dorsolateral PFC (DLPFC) and anterior cingulate cortex (ACC). The levels of nNOS, eNOS and iNOS mRNA were measured by immunocytochemistry and image analysis software in ACC. The co-localization of nNOS and glutamate decarboxylase (GAD65/67) was observed by immunofluorescence double labeling technique. In addition, the selective inhibitor of nNOS, 7-nitroindazole (7-NI), was used to treat ACC slices of mice. The changes in electrophysiological activity of GABA neurons were observed.
Results: The level of CSF-NOx in depression group was significantly lower than that in control group (P = 0.007). In ACC, but not DLPFC, the level of nNOS-mRNA was decreased (P = 0.083). The nNOS-IR kingdom was distributed in layer II/III of ACC in normal human brain. In depression group, the cell density and average optical density of ACC-nNOS-IR in the whole gray matter were decreased (P = 0.083). In control group, the cell density of nNOS-IR in ACC gray matter was positively correlated with CSF-NOx level (rho = 0.667, P = 0.050, n = 9). There was significant co-localization between nNOS and GABAergic neuron marker GAD65/67 in ACC of depressive patients. Slice incubation significantly increased the frequency of GABA-mediated inhibitory postsynaptic currents (mIPSCs) (P 0.05, n=11), but did not change the amplitude of mIPSCs.
CONCLUSION: The expression of ACC-nNOS is down-regulated and the level of CSF-NOx is down-regulated in depressed patients. Selective inhibition of nNOS activity in ACC of mice significantly affects GABAergic neurotransmission.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.4
【引证文献】
相关硕士学位论文 前1条
1 赵玲茏;补心宁神法治疗心胆气虚型惊恐障碍近期临床疗效观察[D];北京中医药大学;2014年
，本文编号：2218840
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