海马齿状回的γ-氨基丁酸对血管性痴呆大鼠空间学习记忆的作用

发布时间：2018-08-13 11:03

【摘要】：血管性痴呆(Vascular Dementia,VD)是继阿尔茨海默病之后第二种最为常见的与年龄相关的神经功能障碍。VD的主要症状是由于脑血管损伤诱发并累积脑组织损伤而导致的渐进性认知功能障碍。学习和记忆是各种认知功能形成的基础,是研究VD认知功能障碍的必选指标。海马是学习记忆的关键部位,尤其在空间学习记忆中起重要作用。哺乳类海马主要分为CA1、CA3和齿状回(dentate gyrus,DG),而DG作为信息进入海马的传入点,对空间信息进行处理和编码,并在空间学习记忆中起着至关重要的作用。虽然研究表明,海马DG区在VD空间学习记忆损害中可能起重要作用,但对参与VD空间记忆损害过程的DG内神经递质及其作用目前尚未完全弄清楚。Y-氨基丁酸(γ-aminobutyricacid,GABA)作为脑内主要的抑制性神经递质,广泛存在于中枢神经系统中,而海马DG内也存在大量的GABA能神经元及其GABAA和GABAB受体。虽然研究表明,DG内的GABA及其GABAA/GABAB受体参与调节突触可塑性及某些学习记忆过程,但其在VD空间学习记忆损害中的作用未见报道。因此,本实验利用双侧颈总动脉永久性结扎法制备VD模型,并应用脑部微量透析法,高效液相色谱分析法,Morris水迷宫(Morris water maze,MWM),免疫组织化学法等实验方法探讨海马DG区GABA及其受体在血管性痴呆模型大鼠空间学习记忆损害中的作用及其部分机制。本实验的具体内容如下:1.利用双侧颈总动脉结扎方法,制备VD模型大鼠,应用MWM观察其空间学习记忆能力、利用脑部微量透析法和高效液相色谱法测定DG区细胞外液中的GABA含量、利用免疫组织化学染色法观察DG区GABAA和GABAB受体表达。2.利用脑部微量注射法往海马DG区注射GABAA和GABAB受体阻断剂观察其对VD模型大鼠空间学习记忆的影响。3.利用脑部微量注射法往海马DG区注射GABAA和GABAB受体阻断剂观察其对VD模型大鼠DG区天冬氨酸(asparate,Asp)、谷氨酸(glutamate,Glu)、谷氨酰胺(glutamine,G1n)、甘氨酸(glycin,Gly)和牛磺酸(taurine,Tau)含量的影响。4.观察正常大鼠海马DG内的GABA在MWM实验过程中的变化。5.观察正常大鼠海马DG区给予GABAA和GABAB受体激动剂对空间学习记忆能力的影响。本实验的结果如下:1.在4d的MWM定位航行实验中,大鼠找到站台所需的逃避潜伏期均随着训练天数的增加而逐渐缩短(均为P0.05),但VD模型组的逃避潜伏期明显长于假手术组(P0.05);训练开始第5天进行空间探索实验,VD模型组穿越原平台区的次数明显少于假手术组(P0.05)。2.VD大鼠海马DG区细胞外液中的GABA浓度为0.78±0.11μM,与假手术组(0.15±0.01μM)相比,GABA的浓度明显增加(P0.05)。3.与假手术组相比,VD大鼠海马DG区GABAA受体的表达没有明显变化(P0.05),而GABAB受体表达显著减少(P0.05)。4.每天训练前往VD大鼠海马DG区微量注射1μl的GABAA受体阻断剂Bicuculline,不影响MWM实验中的逃避潜伏期和穿越原站台区次数(均为P0.05)。5.每天训练前往VD大鼠海马DG区微量注射1μl的GABAB受体阻断剂Saclofen,明显缩短MWM定位航行实验中的逃避潜伏期,并增加MWM空间探索实验中的穿越原站台区的次数(均为P0.05)。6.假手术组大鼠DG区的Glu、G1n和Gly在Morris水迷宫训练过程中明显增加(均为P0.05),而VD模型大鼠DG区的Glu和G1n虽有所增加,但增加明显迟于假手术组,且DG区的Gly在训练过程中无明显变化。7.每天训练前往VD大鼠海马DG区微量注射lμ1的GABAA受体阻断剂Bicuculline,对MWM实验中海马DG内的Glu、Gln和Gly含量没有产生明显的影响。8.每天训练前往VD大鼠海马DG区微量注射1μl的GABAB受体阻断剂Saclofen,可部分翻转VD对Morris水迷宫训练过程中DG区Glu、G1n以及Gly变化的抑制作用。9.在4d的MWM定位航行实验中,正常大鼠的逃避潜伏期均随训练天数的增加而逐渐缩短(P0.05),而训练第五天的空间探索实验中穿越原站台区的次数为7.50±0.43 times/120s。其间,海马DG区的GABA含量随着训练天数的增加逐渐下降(P0.05)。10.每天训练前向海马DG区微量注射1μl的GABAA受体激动剂Muscimol或GABAB受体激动剂Baclofen,30min后进行MWM实验。在4d的定位航行实验中,对照组大鼠的逃避潜伏期随着训练天数的增加而逐渐减少,但Muscimol组和Baclofen组大鼠的逃避潜伏期没有出现明显的变化,两组的平均逃避潜伏期均明显长于对照组(均为P0.05)。训练第五天的空间探索实验中,Baclofen组的穿越原站台区次数明显低于对照组(P0.05),而Muscimol组的穿越原站台区次数虽然有所下降,但与对照组相比无显著性差异(P0.05)。本实验的结论如下:1.VD模型大鼠空间记忆损害与海马DG区的GABA浓度增加有关。2.VD模型大鼠海马DG区内增加的GABA主要通过GABAB受体参与其空间学习记忆的损害。3.海马DG区的Glu、G1n以及Gly参与大鼠的空间学习记忆过程,而DG内的GABAB受体可能通过抑制这些氨基酸类物质的变化导致VD模型大鼠的空间学习记忆损害。
[Abstract]:Vascular Dementia (VD) is the second most common age-related neurological disorder after Alzheimer's disease. The main symptoms of VD are progressive cognitive impairment caused by cerebrovascular injury and cumulative brain tissue damage. Learning and memory are the basis of various cognitive functions and research. Hippocampus is the key part of learning and memory, especially plays an important role in spatial learning and memory. Mammalian hippocampus is mainly divided into CA1, CA3 and dentate gyrus (DG), and DG as the entry point of information into the hippocampus, processes and encodes spatial information, and plays a role in spatial learning and memory. Although studies have shown that the hippocampal DG region may play an important role in the spatial learning and memory impairment of VD, the neurotransmitters and their roles in DG involved in the spatial memory impairment of VD have not been fully understood. In the central nervous system, a large number of GABAergic neurons and their GABAA and GABAB receptors also exist in the hippocampus of DG. Although studies have shown that GABA and its GABAA/GABAB receptors in DG participate in regulating synaptic plasticity and some learning and memory processes, their roles in VD spatial learning and memory impairment have not been reported. VD model was established by permanent ligation of common artery. The effects of GABA and its receptors in hippocampal DG region on spatial learning and memory impairment in vascular dementia rats were studied by microdialysis, high performance liquid chromatography, Morris water maze (MWM) and immunohistochemistry. The specific contents of the experiment are as follows: 1. VD model rats were made by bilateral common carotid artery ligation. The spatial learning and memory abilities were observed by MWM. The GABA content in extracellular fluid of DG region was determined by brain microdialysis and high performance liquid chromatography. The expression of GABAA and GABAB receptors in DG region was observed by immunohistochemical staining. 2. The effects of GABAA and GABAB receptor blockers on spatial learning and memory of VD model rats were observed by intracerebral injection of GABAA and GABAB receptor blockers into the DG region of the hippocampus. The effects of glutamine, G1n, Gly and taurine (Tau) on spatial learning and memory were observed. 4. The changes of GABA in normal rat hippocampal DG during the MWM experiment were observed. 5. The effects of GABAA and GABAB receptor agonists on spatial learning and memory were observed in normal rat hippocampal DG. The results of this experiment were as follows: 1. The escape latency of VD model group was significantly longer than that of sham operation group (P 0.05), and the number of times that VD model group crossed the original platform area was significantly less than that of sham operation group (P 0.05). The concentration of GABA in extracellular fluid of hippocampal DG region was 0.78 [0.11] mu M. Compared with sham operation group, the concentration of GABA increased significantly (P 0.05). 3. Compared with sham operation group, the expression of GABAA receptor in hippocampal DG region of VD rats did not change significantly (P 0.05), but the expression of GABAB receptor decreased significantly (P 0.05). 4. Bicuculline, a 1-ml GABAA receptor blocker, did not affect the escape latency and the number of times crossing the original platform in the MWM experiment (both P 0.05). 5. The 1-ml Saclofen, a GABAB receptor blocker, was microinjected into the DG area of the hippocampus of VD rats every day, which significantly shortened the escape latency in the MWM navigation experiment and increased the spatial exploration of MWM. Glu, G1n and Gly in the DG area of sham-operated rats increased significantly during the training of Morris water maze (all P 0.05). Glu and G1n in the DG area of VD model rats increased slightly, but the increase was significantly later than that of sham-operated rats, and Gly in the DG area did not change significantly during the training. Microinjection of 1-mu-1 GABAA receptor blocker Bicuculline into the DG area of the hippocampus of VD rats had no significant effect on the contents of Glu, Gln and Gly in the DG area of the hippocampus in MWM experiment. The escape latency of normal rats was gradually shortened with the increase of training days (P 0.05), while the number of crossing the original platform was 7.50 (+ 0.43 times / 120 s) in the space exploration experiment on the fifth day of training. MWM test was performed 30 minutes after microinjection of 1 ml of GABAA receptor agonist Muscimol or GABAB receptor agonist Baclofen into the DG area of the hippocampus before training. The escape latency of the control group decreased gradually with the increase of training days, but the escape latency of the Muscimol and Baclofen groups decreased gradually. The average escape latency of the two groups was significantly longer than that of the control group (both P 0.05). In the space exploration experiment on the fifth day of training, the number of crossing the original platform area in the Baclofen group was significantly lower than that in the control group (P 0.05), while the number of crossing the original platform area in the Muscimol group decreased, but compared with the control group. There was no significant difference (P 0.05). The results of this study were as follows: 1. The spatial memory impairment of VD model rats was related to the increase of GABA concentration in hippocampal DG area. 2. The increased GABA in hippocampal DG area of VD model rats participated in the spatial learning and memory impairment mainly through GABAB receptors. 3. Glu, G1n and Gly in hippocampal DG area participated in the spatial learning and memory process of rats. GABAB receptors in DG may cause spatial learning and memory impairment in VD rats by inhibiting the changes of these amino acids.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.13

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