链脲佐菌素损害大鼠条件化恐惧记忆巩固与重建

发布时间：2018-04-20 17:26

本文选题：侧脑室注射 + 散发性阿尔茨海默病　；参考：《南华大学》2013年硕士论文

【摘要】：目的：链脲佐菌素（Streptozotocin，STZ）是一种葡萄糖胺-亚硝基脲衍生物，侧脑室微注射STZ可作为研究散发性阿尔茨海默病（sporadicAlzheimer's disease，SAD）的有效动物模型。在该模型中，STZ可引起长效的大脑葡萄糖代谢和能量代谢损害，同时伴有学习记忆的进展性损伤，如海马依赖的空间学习记忆能力受损。然而，目前仍不清楚STZ诱导的SAD模型动物是否伴有情绪相关的学习记忆能力的损伤。本研究基于大鼠条件化恐惧范式检测了侧脑室微注射STZ对大鼠情绪相关的学习记忆能力的影响，并对其可能的机制进行了初步探讨。方法： 1.大鼠麻醉后经双侧侧脑室微量注射STZ（3mg/kg），48小时后重复注射1次；对照组大鼠微量注射同体积的柠檬酸缓冲液。第一次微注射前1天和第一次微注射后第10天检测外周血糖浓度。 2. Morris水迷宫检测：第一次STZ微注射后第15天进行Morris水迷宫检测，行为训练程序包括两个阶段：（1）定位航行实验，持续4天，每天训练4次，分别从4个不同的位置入水；记录大鼠到达平台的潜伏期。（2）空间搜索实验，第5天撤去平台，大鼠从原始平台位置的对侧入水，记录其在目标象限停留时间。 3.场景性条件化恐惧反应检测：第一次STZ微注射后第15天进行场景性条件化恐惧反应检测，包括习惯化、恐惧条件化、消退、重建和重建检测等五个训练阶段。第1天进行习惯化训练，将大鼠放入A实验箱自由活动10分钟。第2天进行恐惧条件化训练，大鼠在A实验箱中接受5次足底电刺激训练。第3天开始进行恐惧消退训练，持续4天，将大鼠放入A实验箱中20分钟，无足底电刺激。第7天给予2次足底电刺激进行重建训练。第8天进行重建检测。条件化恐惧反应检测指标为场景诱发的大鼠僵直时间。 4.线索性条件化恐惧反应检测：第一次STZ微注射后第14天进行线索性条件化恐惧反应检测，包括习惯化、恐惧条件化、消退、重建和重建检测等五个训练阶段。第1天进行习惯化训练，将大鼠分别放入A和B实验箱内自由活动10分钟。第2天进行恐惧条件化训练，大鼠在A实验箱中接受5次声刺激与足底电刺激配对训练，声刺激与足底电刺激同时结束。第3天开始进行恐惧消退训练，持续3天，每天将大鼠放入B实验箱中单独呈现12次声刺激，无足底电刺激。第6天给予2次单独足底电刺激进行重建训练。第7天进行重建检测即将大鼠放入B实验箱中单独呈现12次声刺激。条件化恐惧反应检测指标为声刺激（线索）诱发的大鼠僵直时间。 5.自发活动检测：第一次STZ微注射后第14天进行自发活动检测，将大鼠放入自发活动检测箱内自由活动30分钟，记录大鼠在此期间的水平活动距离。 6.足底电击敏感性检测：线索性条件化恐惧反应检测结束后第2天进行足底电击敏感性检测，将大鼠放入恐惧条件化实验箱内，记录大鼠开始出现注意朝向反应、退缩反应与惊叫反应时的最小电流强度。 7.高架十字迷宫检测：第一次STZ微注射后第14天进行高架十字迷宫检测，，记录5分钟内大鼠进入开放臂和闭合臂的次数以及在两臂滞留时间。 8.生化检测：行为学检测完成后将大鼠处死取脑，分离海马和杏仁核组织，采用western blot方法检测海马和杏仁核内突触素（Synaptophysin，SYP）、磷酸化环磷腺苷反应元件结合蛋白（phosphorylated cyclicAMP response element binding protein，p-CREB）、总环磷腺苷反应元件结合蛋白（cyclic AMP response element bindingprotein，CREB）和核转录蛋白κB（nuclear transcription factor kappa B，NF-κB）的表达水平。结果： 1. STZ组大鼠外周血糖浓度与对照组比较无显著性组间差异。 2.在Morris水迷宫检测中，STZ组大鼠定位航行实验潜伏期显著性高于对照组；在空间搜索实验中，STZ组大鼠在目标象限停留时间显著性低于对照组。 3.在场景性条件化恐惧反应检测中，STZ组大鼠在第一次消退训练与重建检测阶段其僵直时间百分比与对照组比较均显著性降低；而在其余各训练阶段，两组大鼠僵直时间百分比均无显著性组间差异。 4.在线索性条件化恐惧反应检测中，STZ组大鼠在第一次消退训练与重建检测阶段其僵直时间百分比与对照组比较均显著性降低；而在其余各训练阶段，两组大鼠僵直时间百分比均无显著性组间差异。 5.在自发活动检测中，SZT组大鼠在30分钟内的水平活动距离与对照组比较无显著性组间差异。 6.在足底电击敏感性检测中，SZT组大鼠注意朝向反应、退缩反应与惊叫反应时的最小电流强度与对照组比较均无显著性组间差异。 7.在高架十字迷宫检测中，STZ组大鼠开放臂滞留时间百分比、进入开放臂的次数百分比和进入闭合臂的次数与对照组比较均无显著性组间差异。 8. STZ组大鼠海马和杏仁核内SYP、p-CREB和NF-κB的表达水平均较对照组显著性降低。结论：侧脑室微注射STZ损害大鼠条件化恐惧记忆巩固与重建，其机制可能与STZ诱导海马和杏仁核内SYP、p-CREB和NF-κB水平降低相关。
[Abstract]:Purpose :

In this model , it is not clear whether the induced SAD model is associated with impaired learning and memory ability . However , it is not clear whether the induced SAD model is associated with emotion - related learning and memory ability . However , it is still unclear whether the induced SAD model is associated with emotion - related learning and memory ability .

Method :

1 . After the rats were anesthetized , the rats were injected ( 3 mg / kg ) by a micro - injection at the bilateral lateral ventricle , and the injection was repeated once after 48 hours ;
The concentration of peripheral blood sugar was detected on the first day before the first micro - injection and 10 days after the first micro - injection .

2 . Morris water maze test : the Morris water maze test was performed on the 15th day after the first injection , and the behavior training program consisted of two stages : ( 1 ) positioning the navigation experiment , lasting for 4 days , training four times daily , respectively entering water from four different positions ;
The latency of the rat ' s arrival on the platform was recorded . ( 2 ) Space search experiments , the platform was removed on Day 5 , the rats were fed water from the contralateral side of the original platform position and recorded in the target quadrant residence time .

3 . Detection of Scene Conditional Fear Response : The first day , 15 days after the mini - injection , the rats were subjected to five training stages , such as habit , fear , regression , reconstruction and reconstruction . On the first day , the rats were trained and the rats were placed in A test box for 10 minutes . On day 2 , the rats were placed in A test box for 10 minutes . After the third day , the rats were put into A test box for 20 minutes without plantar electrical stimulation . The reconstruction was performed on the 8th day .

4 . Lead - induced conditioned fear response test : After the first injection , the rats were subjected to five training stages , such as habit , fear , regression , reconstruction and reconstruction . On Day 1 , the rats were placed in A and B experiment boxes for free movement for 10 minutes . After 3 days , the rats were placed in B test box for 12 times of acoustic stimulation .

5 . spontaneous activity detection : spontaneous activity detection was performed on day 14 after the first mini - injection , and the rats were placed in the spontaneous activity detection box for free activity for 30 minutes to record the horizontal movement distance during the period of the rats .

6 . Sensitivity test of foot - bottom electric shock : The sensitivity of plantar electric shock was detected on the second day after the detection of the response of the response of the clue condition , and the rats were put into the experiment box of fear condition , and the minimal current intensity in the reaction , the withdrawal reaction and the panic reaction began to appear in the recording rats .

7 . Elevated cross - maze test : A high - rack cross - maze test was performed on day 14 after the first mini - injection , and the number of rats entering the open arm and the closed arm in 5 minutes was recorded and the residence time of the two arms was recorded .

8 . Biochemical detection : After the completion of the behavioral study , the rats were sacrificed to remove the brain , separate the hippocampus and the amylic nucleus , and the expression level of Synaptophysin ( SYP ) , phosphorylated cyclic AMP response element binding protein ( p - CREB ) , cyclic AMP response element binding protein ( CREB ) and nuclear transcription factor kappa B ( NF - 魏B ) were detected by western blot .

Results :

1 . There was no significant difference between the peripheral blood sugar concentration and the control group .

2 . In Morris water maze test , the latent period of the rats ' positioning navigation was significantly higher than that in the control group .
In the space search experiment , the retention time of the rats in the target quadrant was significantly lower than that in the control group .

3 . The percentage of stiffness and the percentage of stiffness in the control group were significantly lower than those in the control group during the first time of regression training and reconstruction .
There was no significant difference between the two groups during the training period .

4 . Compared with the control group , the percentage of stiffness and the percentage of the control group were significantly lower than those in the control group .
There was no significant difference between the two groups during the training period .

5 . In spontaneous activity test , there was no significant difference in the level of activity between SZT group and control group within 30 minutes .

6 . In the detection of electroshock sensitivity of foot bottom , the rats of SZT group showed no significant difference between the minimum current intensity and the control group when the response was observed .

7 . There was no significant difference between the percentage of open arms and the number of open arms and the number of open arms and the number of closed arms compared with the control group .

8 . The expression levels of SYP , p - CREB and NF - 魏B were significantly lower in the hippocampus and almond nuclei than in the control group .

Conclusion :

The mechanism may be related to the decrease of SYP , p - CREB and NF - 魏B levels in the hippocampus and in the apricot kernel .

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

【共引文献】