海马Racl活性与场景恐惧记忆的维持及遗忘
发布时间：2018-08-23 19:05
【摘要】：目的： 研究海马Racl活性与场景恐惧记忆保持及遗忘的关系,探讨PTSD病理性恐惧记忆不能遗忘的分子机制,为PTSD患者的治疗提供新的理论指导。 方法： 实验一：确定一个能诱导出相似学习曲线,但产生不同恐惧记忆的实验方案。成年健康雄性SD大鼠在条件恐惧操作箱内接受5次电击强度为0.5、0.8或1.2mA的足底电击,两次电击间隔时间为12s、122s或600s,24h后进行恐惧记忆的检测。 实验二：探讨0.8mA电击强度学习后不同时间点产生的恐惧记忆及其与海马Racl活性的关系。动物在条件恐惧操作箱内接受5次电击强度为0.8mA的足底电击,两次电击间隔时间为12s,122s或600s,1h、24h或7d后进行恐惧记忆的检测,部分大鼠电击结束后取材用于Rac-GTP的western blotting (WB)检测及免疫组织化学(IF)染色。 实验三：探讨Racl的活性是否调节着大鼠的场景恐惧记忆。成年雄性SD大鼠在条件恐惧操作箱内接受5次足底电击,两次电击问隔时间为12s或122s,12s组大鼠电击结束后立即双侧海马CAl区予Racl抑制剂NSC23677,122s组大鼠双侧海马CA1区予Racl激动剂CN04-A后行足底电击,24h和(或)7天后进行恐惧记忆的检测。 实验四：探讨场景恐惧记忆的消退与海马Racl活性的关系。成年雄性SD大鼠在条件恐惧操作箱内接受5次足底电击,24h后在条件恐惧操作箱内接受6次每次5min的消退训练,两次消退训练间隔时间为0min,10min或30min,消退结束后24h和16d进行恐惧消退的检测。部分大鼠消退结束后1h取材用于WB或IF。 实验五：探讨Racl活性是否直接调节场景恐惧记忆的消退。成年雄性SD大鼠在条件恐惧操作箱内接受5次足底电击,24h后在条件恐惧操作箱内接受6次每次5min消退训练,两次消退训练无间隔,消退训练结束后立即双侧海马CA1区予Racl抑制剂NSC23677,24h和16d后进行恐惧记忆的检测。 实验六：进一步探讨Racl活性是否调节着场景恐惧记忆的消退。成年雄性SD大鼠在条件恐惧操作箱内接受5次足底电击,24h后接受6次每次10min的强消退训练,两次消退训练间隔时间为0min,10min或30min,消退结束后24h和16d进行恐惧记忆的检测；部分大鼠消退结束后1h取材用于WB或IF。结果： 实验一：0.5mA电击强度学习后,三组大鼠(12s,122s和600s)24小时恐惧记忆检测无明显差异,0.8mA或1.2mA电击强度学习后,122s和600s组大鼠表现为场景恐惧记忆增强,与12s组比具有统计学差异,此结果显示0.8mA符合我们的实验要求。 实验二：学习后1小时恐惧记忆检测结果显示122s和600s组大鼠恐惧记忆明显强于12s组,具有统计学差异；24h、7d恐惧记忆检测结果类似1h,122s和600s组大鼠恐惧记忆显著强于12s组。Western blot检测结果显示条件恐惧学习后0h时间点三组大鼠海马Rac1-GTP的表达与正常对照大鼠相似；1h时间点间隔学习组(122s和600s组)大鼠海马Rac1-GTP的表达显著下调,其中122s组低于600s组,集中学习组(12s)Rac1-GTP的表达类似正常组；24h时间点间隔学习组(122s和600s组)大鼠海马Rac1-GTP的表达仍下调,但与正常对照相比未达统计学差异,集中学习组Rac1-GTP的表达类似正常组。免疫荧光染色结果显示正常大鼠Rac1-GTP的染色主要分布于神经元的胞膜和突起,可见Rac1-GTP阳性的胶质细胞及中间神经元。集中学习组海马CA1区Rac1-GTP的染色类似正常组,间隔学习组(122s和600s组)CA1区神经元Rac1-GTP染色明显减弱。此结果显示间隔学习诱发海马Racl活性下调及强的场景恐惧记忆。 实验三：24h恐惧记忆检测结果显示予Racl抑制剂(12s+NSC)组大鼠恐惧记忆明显强于予生理盐水(12s+sal)组,达到122s组水平,差异具有显著性,7d再次检测结果类似24h。恐惧学习后7d第一次行恐惧记忆检测的结果同样显示Racl抑制剂(12s+NSC)组大鼠恐惧记忆明显高于生理盐水对照(12s+sal)组,达统计学差异；Racl激动剂CN04-A (122s+CN04-A)组大鼠24h恐惧记忆检测显著低于生理盐水对照组(122s+sal),7d再次检测结果类似24h。此结果显示调节海马Racl活性可调节大鼠场景恐惧记忆的强弱。 实验四：结果显示0min组(集中消退)恐惧消退训练时成绩明显优于10min和30min组(间隔消退),消退训练后24h检测结果显示三组大鼠(0min、10min、30min)恐惧记忆均出现明显消退,组间无差异；消退后16d再次检测结果显示0min组大鼠恐惧记忆进一步降低,10min组部分恢复,30min组几乎完全恢复,三组间达统计学差异。WB检测结果显示0min组大鼠海马Racl-GTP表达显著上调,30mmin组轻度下调；免疫荧光染色结果显示0min组大鼠海马CA1区Rac1-GTP染色增强,30min组染色减弱。此结果显示集中消退促进大鼠海马Racl的激活及明显的场景恐惧记忆消退。 实验五：24h恐惧消退结果显示Racl抑制剂(0min+NSC)组大鼠恐惧记忆高于生理盐水组(0min+sal),但未达统计学差异,16d再次检测结果显示Racl抑制剂(0min+NSC)组大鼠恐惧记忆明显高于生理盐水(0min+sal)组,两组间具有统计学差异。此结果显示抑制海马Rac l的激活清楚了集中消退诱发的场景恐惧记忆较弱效应。 实验六：结果显示0min组(集中消退)大鼠消退训练时消退成绩明显优于10min和30min组(间隔消退),消退训练后24h检测结果显示三组大鼠(0min、10min、30min)恐惧记忆均出现明显消退,其中10min组略高于0min组和30min组,但组问无统计学差异；消退后16d检测结果显示三组大鼠的恐惧记忆均出现明显消退。WB检测结果显示与正常对照组相比,0min和10min组大鼠海马Racl-GTP的表达均上调,其中0min组达统计学差异。免疫荧光染色显示0min和10min组大鼠海马CA1区Racl-GTP染色均增强,30min组染色与正常对照组相似。此结果显示促进海马Racl活性的恢复促进了场景恐惧记忆的消退。结论： 抑制海马Racl活性导致大鼠场景恐惧记忆增强及场景恐惧记忆消退受损,激活海马Racl致使大鼠场景恐惧记忆减弱及场景恐惧记忆消退增加,海马Racl活性水平调节着场景条件恐惧学习大鼠场景恐惧记忆的强弱及场景恐惧记忆的消退效果。Racl活性调节着果蝇记忆的遗忘,因此,我们提出海马Racl的活性调节着大鼠场景恐惧记忆的维持与遗忘,本研究提示PTSD患者病理性恐惧记忆增强可能与其海马Racl不能正常激活,导致恐惧记忆的遗忘受损有关。
[Abstract]:Objective:
To study the relationship between the activity of Racl in hippocampus and the retention and forgetting of scene fear memory, and to explore the molecular mechanism of PTSD pathological fear memory.
Method:
Experiment 1: To determine an experimental scheme which can induce similar learning curve but produce different fear memory. Adult healthy male SD rats were subjected to 5 plantar shocks of 0.5, 0.8 or 1.2 mA in the conditioned fear operating box. The interval between the two shocks was 12 s, 122 s or 600 s, and the fear memory was tested 24 hours later.
Experiment 2: To explore the relationship between fear memory and Racl activity in the hippocampus at different time points after 0.8 mA shock intensity learning. After completion, Western blotting (WB) detection and immunohistochemical staining (IF) were applied to Rac-GTP.
Experiment 3: To investigate whether the activity of Racl regulates the memory of scene fear in rats. Adult male SD rats received 5 plantar shocks in the conditioned fear operating box. The interval time between the two shocks was 12 s or 122 s. Rats in the 12s group were given Racl stimulation in bilateral hippocampal CAl area immediately after the shock. After CN04-A, the foot was electrocuted, and 24h and (or) 7 days later, the fear memory was detected.
Experiment 4: To investigate the relationship between the deterioration of scene fear memory and the activity of Racl in hippocampus.Adult male SD rats were subjected to five plantar shocks in the conditioned fear operating box. After 24 hours, they were subjected to six 5-minute withdrawal training sessions in the conditioned fear operating box. The intervals of the two withdrawal training sessions were 0 minutes, 10 minutes or 30 minutes. After the recession, they were terrorized for 24 hours and 16 days. Detection of fear extinction. After withdrawal of some rats, 1H was taken for WB or IF..
Experiment 5: To investigate whether Racl activity directly regulates the decline of scene fear memory. Adult male SD rats received five plantar shocks in the conditioned fear operating box. After 24 hours, they received six 5-minute regression training sessions in the conditioned fear operating box. There was no interval between the two regression training sessions. NSC23677,24h and 16d were used to detect fear memory.
Experiment 6: To further explore whether Racl activity regulates the decline of scene fear memory. Adult male SD rats were subjected to five plantar shocks in the conditioned fear manipulation box and six 10-minute intensive regression training sessions after 24 hours. The intervals of the two regression training sessions were 0 minutes, 10 minutes or 30 minutes. Fear memory was tested 24 hours and 16 days after the regression. After the extinction of some rats, 1H was taken for WB or IF..
Experiment 1: After 0.5mA shock intensity learning, there was no significant difference in 24-hour fear memory test among the three groups (12s, 122s and 600s). After 0.8mA or 1.2mA shock intensity learning, the rats in 122s and 600s groups showed enhanced scene fear memory, which was significantly different from that in 12S group. The results showed that 0.8mA met our experimental requirements.
Experiment 2: The results of fear memory test showed that the fear memory of rats in 122s and 600s groups was significantly stronger than that of 12S group, and the difference was statistically significant. The results of fear memory test in 24h and 7d groups were similar to that in 1h, 122s and 600s groups were significantly stronger than that in 12S group. Rac1-GTP expression in hippocampus was similar to that in normal control rats; Rac1-GTP expression in hippocampus was significantly down-regulated in 1-hour interval learning group (122 s and 600 s groups), of which 122 s group was lower than 600 s group, and Rac1-GTP expression in concentrated learning group (12s) was similar to that in normal group; Rac1-GTP expression in hippocampus was still down-regulated in 24-hour interval learning group (122 s and 600 s groups). The expression of Rac1-GTP in the central learning group was similar to that in the normal control group. Immunofluorescence staining showed that Rac1-GTP staining was mainly distributed in the membranes and processes of neurons, and Rac1-GTP positive glial cells and intermediate neurons were observed. Rac1-GTP staining in the hippocampal CA1 region of the central learning group was observed. Similar to the normal group, the Rac1-GTP staining of CA1 neurons in the interval learning group (122s and 600s) was significantly decreased. The results showed that interval learning induced a decrease in Racl activity in the hippocampus and a strong memory of scene fear.
Experiment 3: The results of 24-hour fear memory test showed that the fear memory of rats treated with Racl inhibitor (12s + NSC) was significantly stronger than that of rats treated with normal saline (12s + sal), and the difference was significant. The results of 7-day repeat test were similar to those of 24-hour repeat test. Fear memory of rats was significantly higher than that of normal saline group (12s+sal), and the fear memory of rats in Racl agonist CN04-A (122s+CN04-A) group was significantly lower than that of normal saline group (122s+sal) at 24h, and the result of repeat test was similar to that of normal saline group (122s+sal) at 7d.
Experiment 4: The results showed that the scores of fear subsidence training in 0-minute group were significantly better than those in 10-minute and 30-minute groups (interval subsidence). The results of 24 hours after subsidence training showed that the fear memory of the three groups of rats (0, 10, 30 minutes) had evident subsidence, and there was no difference between the three groups 16 days after subsidence. WB test showed that the expression of Racl-GTP in the hippocampus of rats in 0 min group was significantly up-regulated, but slightly down-regulated in 30 mmin group. Immunofluorescence staining showed that the expression of Rac1-GTP in CA1 area in the hippocampus of rats in 0 min group was enhanced and the staining was weakened in 30 min group. Regression promoted the activation of hippocampal Racl and the extinction of scene fear memory in rats.
Experiment 5: The results of 24-hour fear subsidence showed that the fear memory of rats in Racl inhibitor group (0 min + NSC) was higher than that of normal saline group (0 min + sal), but there was no significant difference. The results of 16-day re-test showed that the fear memory of rats in Racl inhibitor group (0 min + NSC) was significantly higher than that of normal saline group (0 min + sal). The activation of Rac L in hippocampus made clear the weak effect of fear of memory evoked by concentrated regression.
Experiment 6: The results showed that the extinction performance of 0-minute group was significantly better than that of 10-minute and 30-minute group (interval extinction). The results of 24 hours after extinction training showed that the fear memory of the three groups of rats (0, 10, 30 minutes) had evident extinction, of which 10-minute group was slightly higher than that of 0-minute group and 30-minute group, but there was no significant difference between the two groups. WB test showed that the expression of Racl-GTP in the hippocampus of rats in 0 min and 10 min groups was up-regulated compared with the normal control group, and the expression of Racl-GTP in the hippocampus of rats in 0 min and 10 min groups was statistically different. Immunofluorescence staining showed that the expression of Racl-GTP in the CA1 area of the hippocampus of rats in 0 min and 10 min groups was enhanced, and that in 30 min group was up-regulated. Similar to the normal control group, this result showed that the recovery of Racl activity in hippocampus promoted the decline of scene fear memory.
Inhibition of Racl activity in hippocampus leads to the enhancement of scene fear memory and the deterioration of scene fear memory in rats. Activation of Racl in hippocampus leads to the decrease of scene fear memory and the increase of scene fear memory in rats. Racl activity in hippocampus regulates the intensity of scene fear memory and the deterioration of scene fear memory in scene conditioned fear learning rats. Therefore, we suggest that the activity of hippocampal Racl regulates the maintenance and forgetting of scene fear memory in rats. This study suggests that the enhancement of pathological fear memory in PTSD patients may be related to the inability of hippocampal Racl to activate normally, resulting in impaired forgetting of fear memory.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.7
[Abstract]:Objective:
To study the relationship between the activity of Racl in hippocampus and the retention and forgetting of scene fear memory, and to explore the molecular mechanism of PTSD pathological fear memory.
Method:
Experiment 1: To determine an experimental scheme which can induce similar learning curve but produce different fear memory. Adult healthy male SD rats were subjected to 5 plantar shocks of 0.5, 0.8 or 1.2 mA in the conditioned fear operating box. The interval between the two shocks was 12 s, 122 s or 600 s, and the fear memory was tested 24 hours later.
Experiment 2: To explore the relationship between fear memory and Racl activity in the hippocampus at different time points after 0.8 mA shock intensity learning. After completion, Western blotting (WB) detection and immunohistochemical staining (IF) were applied to Rac-GTP.
Experiment 3: To investigate whether the activity of Racl regulates the memory of scene fear in rats. Adult male SD rats received 5 plantar shocks in the conditioned fear operating box. The interval time between the two shocks was 12 s or 122 s. Rats in the 12s group were given Racl stimulation in bilateral hippocampal CAl area immediately after the shock. After CN04-A, the foot was electrocuted, and 24h and (or) 7 days later, the fear memory was detected.
Experiment 4: To investigate the relationship between the deterioration of scene fear memory and the activity of Racl in hippocampus.Adult male SD rats were subjected to five plantar shocks in the conditioned fear operating box. After 24 hours, they were subjected to six 5-minute withdrawal training sessions in the conditioned fear operating box. The intervals of the two withdrawal training sessions were 0 minutes, 10 minutes or 30 minutes. After the recession, they were terrorized for 24 hours and 16 days. Detection of fear extinction. After withdrawal of some rats, 1H was taken for WB or IF..
Experiment 5: To investigate whether Racl activity directly regulates the decline of scene fear memory. Adult male SD rats received five plantar shocks in the conditioned fear operating box. After 24 hours, they received six 5-minute regression training sessions in the conditioned fear operating box. There was no interval between the two regression training sessions. NSC23677,24h and 16d were used to detect fear memory.
Experiment 6: To further explore whether Racl activity regulates the decline of scene fear memory. Adult male SD rats were subjected to five plantar shocks in the conditioned fear manipulation box and six 10-minute intensive regression training sessions after 24 hours. The intervals of the two regression training sessions were 0 minutes, 10 minutes or 30 minutes. Fear memory was tested 24 hours and 16 days after the regression. After the extinction of some rats, 1H was taken for WB or IF..
Experiment 1: After 0.5mA shock intensity learning, there was no significant difference in 24-hour fear memory test among the three groups (12s, 122s and 600s). After 0.8mA or 1.2mA shock intensity learning, the rats in 122s and 600s groups showed enhanced scene fear memory, which was significantly different from that in 12S group. The results showed that 0.8mA met our experimental requirements.
Experiment 2: The results of fear memory test showed that the fear memory of rats in 122s and 600s groups was significantly stronger than that of 12S group, and the difference was statistically significant. The results of fear memory test in 24h and 7d groups were similar to that in 1h, 122s and 600s groups were significantly stronger than that in 12S group. Rac1-GTP expression in hippocampus was similar to that in normal control rats; Rac1-GTP expression in hippocampus was significantly down-regulated in 1-hour interval learning group (122 s and 600 s groups), of which 122 s group was lower than 600 s group, and Rac1-GTP expression in concentrated learning group (12s) was similar to that in normal group; Rac1-GTP expression in hippocampus was still down-regulated in 24-hour interval learning group (122 s and 600 s groups). The expression of Rac1-GTP in the central learning group was similar to that in the normal control group. Immunofluorescence staining showed that Rac1-GTP staining was mainly distributed in the membranes and processes of neurons, and Rac1-GTP positive glial cells and intermediate neurons were observed. Rac1-GTP staining in the hippocampal CA1 region of the central learning group was observed. Similar to the normal group, the Rac1-GTP staining of CA1 neurons in the interval learning group (122s and 600s) was significantly decreased. The results showed that interval learning induced a decrease in Racl activity in the hippocampus and a strong memory of scene fear.
Experiment 3: The results of 24-hour fear memory test showed that the fear memory of rats treated with Racl inhibitor (12s + NSC) was significantly stronger than that of rats treated with normal saline (12s + sal), and the difference was significant. The results of 7-day repeat test were similar to those of 24-hour repeat test. Fear memory of rats was significantly higher than that of normal saline group (12s+sal), and the fear memory of rats in Racl agonist CN04-A (122s+CN04-A) group was significantly lower than that of normal saline group (122s+sal) at 24h, and the result of repeat test was similar to that of normal saline group (122s+sal) at 7d.
Experiment 4: The results showed that the scores of fear subsidence training in 0-minute group were significantly better than those in 10-minute and 30-minute groups (interval subsidence). The results of 24 hours after subsidence training showed that the fear memory of the three groups of rats (0, 10, 30 minutes) had evident subsidence, and there was no difference between the three groups 16 days after subsidence. WB test showed that the expression of Racl-GTP in the hippocampus of rats in 0 min group was significantly up-regulated, but slightly down-regulated in 30 mmin group. Immunofluorescence staining showed that the expression of Rac1-GTP in CA1 area in the hippocampus of rats in 0 min group was enhanced and the staining was weakened in 30 min group. Regression promoted the activation of hippocampal Racl and the extinction of scene fear memory in rats.
Experiment 5: The results of 24-hour fear subsidence showed that the fear memory of rats in Racl inhibitor group (0 min + NSC) was higher than that of normal saline group (0 min + sal), but there was no significant difference. The results of 16-day re-test showed that the fear memory of rats in Racl inhibitor group (0 min + NSC) was significantly higher than that of normal saline group (0 min + sal). The activation of Rac L in hippocampus made clear the weak effect of fear of memory evoked by concentrated regression.
Experiment 6: The results showed that the extinction performance of 0-minute group was significantly better than that of 10-minute and 30-minute group (interval extinction). The results of 24 hours after extinction training showed that the fear memory of the three groups of rats (0, 10, 30 minutes) had evident extinction, of which 10-minute group was slightly higher than that of 0-minute group and 30-minute group, but there was no significant difference between the two groups. WB test showed that the expression of Racl-GTP in the hippocampus of rats in 0 min and 10 min groups was up-regulated compared with the normal control group, and the expression of Racl-GTP in the hippocampus of rats in 0 min and 10 min groups was statistically different. Immunofluorescence staining showed that the expression of Racl-GTP in the CA1 area of the hippocampus of rats in 0 min and 10 min groups was enhanced, and that in 30 min group was up-regulated. Similar to the normal control group, this result showed that the recovery of Racl activity in hippocampus promoted the decline of scene fear memory.
Inhibition of Racl activity in hippocampus leads to the enhancement of scene fear memory and the deterioration of scene fear memory in rats. Activation of Racl in hippocampus leads to the decrease of scene fear memory and the increase of scene fear memory in rats. Racl activity in hippocampus regulates the intensity of scene fear memory and the deterioration of scene fear memory in scene conditioned fear learning rats. Therefore, we suggest that the activity of hippocampal Racl regulates the maintenance and forgetting of scene fear memory in rats. This study suggests that the enhancement of pathological fear memory in PTSD patients may be related to the inability of hippocampal Racl to activate normally, resulting in impaired forgetting of fear memory.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R749.7
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