大鼠边缘下区Cdk5及突触结构在条件性恐惧消退训练后的变化研究
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摘要
近些年,各种灾难频繁发生,创伤后应激障碍(post-traumatic stress disorder, PTSD)的发病率有增加的趋势,并且目前对PTSD发病机制的认识还不够深入,导致其治疗存在许多困难,因此PTSD也成为科学家研究的热点。临床上,治疗PTSD的常用方法是暴露疗法,即对创伤记忆进行消退,但已消退的恐惧记忆又容易再现,提示消退记忆保持障碍可能是PTSD发生和治疗困难的关键。以往的研究表明,前额叶皮层(prefrontal cortex, PFC)的功能状态与PTSD的发生与发展密切相关。啮齿类动物PFC的边缘下皮层(infralimbic cortex, IL)则是消退记忆保持的重要部位,然而,关于该区在消退记忆中的分子机制尚不完全清楚。
许多研究显示,细胞周期素依赖性蛋白激酶5(Cyclin-dependent kinase 5, Cdk5)及其激活剂P35广泛存在于大脑皮层,Cdk5/P35复合物可改变海马中突触的功能和结构可塑性,并影响条件性恐惧记忆的形成以及消退记忆的习得。但是,在条件性恐惧消退训练后,大鼠IL区Cdk5和P35的蛋白表达以及激酶活性如何变化?这个过程中IL区的突触结构又会发生怎样的变化?这些均未见报道。为此,我们设计了以下两个实验。
实验一、条件性恐惧消退训练后大鼠边缘下区Cdk5的表达和酶活性的变化
目的:探讨在条件性恐惧消退训练后,大鼠IL区Cdk5表达和活性的变化。方法:将雄性SD大鼠随机分为消退组、消退对照组和正常对照组。采用声音结合足底电击的方式建立条件性恐惧模型,消退组在恐惧建立后24h进行消退训练,消退对照组不进行消退训练,两组均在不同时间点(恐惧建立后第2、4和8天)进行行为学测试后,使用免疫组织化学法检测各组大鼠IL区Cdk5的免疫反应阳性细胞数、Western blot检测Cdk5及其激活剂P35、P25的蛋白表达水平以及液闪法检测Cdk5活性的变化。
结果:(1)消退组和消退对照组的不僵立时间百分比在恐惧建立后第2天和第4天均显著低于正常组(86.03±8.90,P <0.01);到恐惧建立后第8天,消退组基本恢复正常,而消退对照组(36.18±12.60)仍低于正常组(P <0.01)。同组内不同时间点的比较:随时间进展,两实验组的恐惧水平均呈逐渐下降的趋势(P <0.01)。
(2)免疫组化结果显示:在恐惧建立后第2天,消退组大鼠IL区Cdk5的免疫反应阳性细胞数与正常组无差异,消退对照组的阳性细胞数(429±70)却多于正常组(322±52,P =0.026)和消退组(358±63,P <0.05);在恐惧建立后第4天,消退组的阳性细胞数明显减少(208±30,P <0.01),消退对照组基本恢复正常;在恐惧建立后第8天,消退组也恢复正常,与消退对照组无统计学差异。
(3)Western blot的结果为:在消退训练后1周内,消退组大鼠IL区Cdk5的蛋白表达呈减少的趋势:消退后第1和3天的表达与正常组的差异无统计学意义,而到消退后第7天(1.35±0.13)少于正常组(1.89±0.06,P <0.05);消退对照组的Cdk5蛋白表达在恐惧建立后第4天增多(2.94±0.19),明显高于消退组(1.78±0.20,P <0.01)和正常组(P <0.01);到恐惧建立后第8天基本恢复正常,但显著多于消退组(P <0.01)。
两组P35的表达与正常组无显著性差异;而两组P25的蛋白表达均显著高于正常组。在消退训练后1周,消退组P25的表达逐渐减少:消退后第7天(1.60±0.12)明显低于消退后第1天(2.14±0.24,P =0.032)和第3天(2.17±0.21,P =0.026);消退对照组在这个过程中则有增加的趋势:恐惧建立后第2天的表达(1.78±0.16)明显少于恐惧建立后第4天(2.50±0.19,P =0.039)和第8天(2.58±0.40,P =0.027)。
(4)液闪法的结果为:消退组大鼠IL区Cdk5的活性在消退训练后第1和3天显著增加(产物的放射性活度值分别为3107.78±536.42和3330.05±597.82,P <0.01),但到消退后第7天(2179.89±327.20)基本恢复到正常水平(1812.08±175.21);而消退对照组在这三天均明显高于正常组(P <0.05或P <0.01)。两实验组比较显示,在恐惧建立后第2天无统计学差异,消退组的激酶活性在恐惧建立后第4和第8天都显著低于消退对照组(P <0.05或P <0.01)。
结论:条件性恐惧使IL区Cdk5的蛋白表达和活性升高,大鼠的僵立行为较多;而经过消退训练后,Cdk5的蛋白表达逐渐降低、活性较快恢复正常,大鼠的恐惧反应也逐渐减少,这些变化可能有利于消退记忆的保持。
实验二、条件性恐惧消退训练后大鼠边缘下区突触数密度及突触界面结构的变化
目的:探讨条件性恐惧消退训练后,大鼠IL区突触数密度和突触界面结构的变化。
方法:将雄性SD大鼠随机分为消退组、消退对照组和正常对照组。采用声音结合足底电击的方式建立条件性恐惧模型,消退组在恐惧建立后24h进行消退训练,消退对照组不进行消退训练,两组均在不同时间点(恐惧建立后第8和22天)测定大鼠的僵立行为,然后快速断头取IL区按常规方法制作透射电镜标本,并用透射电镜观察、摄像,分析该区突触数密度和突触界面结构参数,包括:突触界面曲率、活性区长度、突触间隙宽度和突触后致密物质(postsynaptic density,PSD)厚度的变化。
结果:(1)行为学:消退组大鼠在两个时间点的保持成绩均与正常组(91.17±3.07)无显著差异;消退对照组在恐惧建立后第22天(66.73±8.75)的不僵立时间多于第8天(38.40±10.12,P =0.021),却都明显少于正常组(P <0.01)和消退组(P <0.05或P <0.01)。
(2)突触数密度:在两个时间点,消退组大鼠IL区的突触数密度(单位体积下的突触数量)均明显高于正常组(0.36±0.06,P <0.05);而消退对照组在恐惧建立后第8天的突触数密度(0.39±0.04)与正常无差异,但在恐惧建立后第22天(0.22±0.03)明显低于正常组(P =0.038)和消退组(0.48±0.06,P <0.01)。
(3)突触界面参数:三组间比较显示,突触界面曲率和突触间隙无统计学差异,而活性区长度和PSD厚度有显著性差异。与正常组相比,在消退训练后第7天,消退组大鼠IL区的PSD厚度无明显变化,到消退后第21天,PSD厚度增加(53.87±11.39,P =0.017);消退对照组则在两个时间点均表现为PSD的厚度变薄(P <0.05或P <0.01),且突触活性区长度均小于消退组(P <0.05或P <0.01)。
结论:条件性恐惧的建立使IL区的突触数密度减少,突触的结构受到损害,而消退训练后,该区的突触数密度增加,突触传递效率增强,有利于条件性恐惧反应的减少。
总之,从这两个实验可以推测:条件性恐惧消退训练后,IL区Cdk5的改变与IL区突触结构可塑性的变化存在一定的联系,即条件性恐惧建立后,大鼠IL区的Cdk5活性和P25表达持续升高,可能通过减少突触密度、改变突触结构,使该区的突触功能下降,大鼠的恐惧反应持续存在;然而,消退训练则使P25的表达逐渐下降、Cdk5活性较快恢复正常,可能有利于突触的结构可塑性,在行为上表现为大鼠的僵立时间百分比能较快恢复正常水平。
In recent years, catastrophic events occur so frequently that the incidence of post-traumatic stress disorder (PTSD) has a gradual growth. whereas, the pathogenesis of PTSD remains elusive, which leads to many difficulties to cure this disease. Recently, more and more scientists concern about PTSD. At present, the exposure therapy is usually used in treatment of PTSD, that is fear extinction, but fear memory easily appears again. This suggests that retention disorder of extinction memory may be a key factor in the occurrence and treatment of PTSD. Previous studies showed that the functional status of prefrontal cortex (PFC) was closely related to the occurrence and development of PTSD, and infralimbic cortex (IL) of PFC in rodents was an important site to store the extinction memory. However, it is not clear which substances involve in the process of retention of extinction memory in IL.
The serine/threonine kinase, cyclin-dependent kinase 5 (Cdk5) and its activator P35, which widespread in the cerebral cortex, could mediate functional and structural plasticity of synapes, and also affect the formation of fear memory and the acquisition of extinction memory. After extinction training, how do the Cdk5 expression and kinase activity change in IL of rats, and what changes will be observed about the synaptic structure in IL area. The present research aimed to answer these questions by two experiments.
The first experiment
Changes of Cdk5 expression and kinase activity in infra-limbic cortex of rats after conditioned fear extinction training
Objective: To investigate the changes of Cdk5 expression and kinase activity in IL area of rats after extinction training.
Methods: Male adult SD rats were randomly divided into extinction group (receiving fear conditioning and extinction training, EXT group), conditioned fear group (receiving fear training but no extinction training,Cont group) and natural comparison group(without any disposal, Naive group). Conditioned fear model of rats was established by six tones (unconditioned stimulus) paired with foot electric shock (conditioned stimulus). The extinction trials, which were given to animals at 24 hours after the last conditioning trail, were 16 tones without electric shock. Freezing behavior, expression and activity of Cdk5 were assessed on different days (2d, 4d and 8d) after fear conditioning.
Results:
(1) The freezing behaviors of EXT group and Cont group were more than naive group (86.03±8.90) on 2d and 4d (P <0.01). Until the 8d after fear conditioning, the freezing score of EXT group declined to the level of naive group, but it was less than that in Cont group (36.18±12.60, P <0.01).
(2) On 2d, the number of immunoreactive positive cells of Cdk5 significantly increased in IL area of Cont group (429±70, P <0.05). On 4d and 8d, there were no disparities between Cont group and naive group. Whereas the number of cells in EXT group decreased on 4d (208±30, P <0.01), but returned to normal on 8d.
(3) Western blot results showed: compared with naive group (1.89±0.06), the expression of Cdk5 in IL area of EXT group had a tendency to decrease, and the expression was lowest on 8d (1.35±0.13, P <0.05). While the expression of Cdk5 in Cont group increased on 4d (2.94±0.19), which was more than that in naive group and EXT group (1.78±0.20, P <0.01), and recovered almost to normal on 8d. No significant differences were found in the expression of P35 among the three groups, but the expression of P25 in experiment groups all were higher than that in naive group. After fear conditioning, the expression of P25 gradully increased, and reached peak on 8d (2.58±0.40,P <0.05). After extinction trials, the expression of P25 gradully decreased, and was lowest on 8d (1.60±0.12,P <0.05).
(4) After fear conditioning, the activity of Cdk5 enhanced. The activities in Cont group were higher than that in naive group (1812.08±175.21, P <0.05 or P <0.01) on 2d, 4d, and 8d. In contrast, although the activities of Cdk5 in EXT group had an obvious increasing on 2d (3107.78±536.42, P <0.01) and 4d (3330.05±597.82, P <0.01), the activity retured to normal level again(2179.89±327.20) on 8d, but was lower than that in Cont group (P <0.01).
Conclusion: After fear conditioning, the expression and activity of Cdk5 in IL area may increase, and the freezing behaviors of rats continue for a long time. But extinction training diminishes its expression and activity, which may be good for the retention of extinction memory, so that the fear response return to normal quickly.
The second experiment
Changes of synaptic numerical density and texture parameters analyses in infra-limbic cortex of rats after conditioned fear extinction training
Objective: To observe the changes of synaptic numerical density and texture parameters in IL area of rats after extinction training.
Methods: Male adult SD rats were randomly divided into EXT group, Cont group and Naive group (natural comparison group). EXT group and Cont group were all given 6 tone-foot shock paired trainings. But extinction trials were only given to EXT group 24 hours after fear conditioning. Behavioral tests were done at different days (8d and 22d) after fear training, and transmission electron microscope was used to observe the synaptic numerical density and texture parameters, including the thickness of postsynaptic density (PSD), the width of synaptic cleft, the curvature of synaptic interface and the length of active zone.
Results:
(1) No statistical differences were observed in freezing behavior between the EXT group and naive group on 8d and 22d. The freezing scores of Cont group had a trend to decrease, but the scores were all higher than that in EXT group and naive group on 8d and 22d (P <0.05 or P <0.01).
(2) The synaptic numerical densities in IL area of EXT group all were more than that in naive group (0.36±0.06, P <0.05) on 8d and 22d. Whereas on 8d after fear conditioning, the synaptic numerical densities in Cont group was equal to that in naive group, and decreased on 22d (0.22±0.03, P <0.05).
(3) The results of texture parameters analysing showed: there were no statistical differences in the curvature of synaptic interface and the width of synaptic cleft among the three groups. Compared with naive group, the thickness of PSD and the length of active zone had not obvious changes in EXT group on 8d, but the PSD became thickening notably on 22d (53.87±11.39, P <0.05). For the Cont group, the thickness of PSD was thinner than that in naive group and EXT group (P <0.05 or P <0.01) on 8d and 22d, and the length of active zone was less than that in EXT group (P <0.05 or P <0.01).
Conclusion: The establishment of the conditioned fear may decrease the synaptic numerical density in IL area of rats, and impair the synaptic structure. However, after extinction training, the synaptic numerical densities increase, and synaptic function could be enhanced, these changes may be good for fear extinction.
From these two experiments, it is possible that there is a relationship between Cdk5 and the changes of synaptic structural plasicity in IL area of rats. After fear conditioning, hyperexpression of P25 sustained more than one week, which caused Cdk5 activity increasing continuely, and could impair the synaptic structural plasticity of IL area. These changes may result in freezing behavior of rats. Through extinction training, the expression of P25 decreased gradually, and the kinase activity retured to normal fastily, which may be beneficial for the synaptic structural plasticity and conditioned fear extinction.
许多研究显示,细胞周期素依赖性蛋白激酶5(Cyclin-dependent kinase 5, Cdk5)及其激活剂P35广泛存在于大脑皮层,Cdk5/P35复合物可改变海马中突触的功能和结构可塑性,并影响条件性恐惧记忆的形成以及消退记忆的习得。但是,在条件性恐惧消退训练后,大鼠IL区Cdk5和P35的蛋白表达以及激酶活性如何变化?这个过程中IL区的突触结构又会发生怎样的变化?这些均未见报道。为此,我们设计了以下两个实验。
实验一、条件性恐惧消退训练后大鼠边缘下区Cdk5的表达和酶活性的变化
目的:探讨在条件性恐惧消退训练后,大鼠IL区Cdk5表达和活性的变化。方法:将雄性SD大鼠随机分为消退组、消退对照组和正常对照组。采用声音结合足底电击的方式建立条件性恐惧模型,消退组在恐惧建立后24h进行消退训练,消退对照组不进行消退训练,两组均在不同时间点(恐惧建立后第2、4和8天)进行行为学测试后,使用免疫组织化学法检测各组大鼠IL区Cdk5的免疫反应阳性细胞数、Western blot检测Cdk5及其激活剂P35、P25的蛋白表达水平以及液闪法检测Cdk5活性的变化。
结果:(1)消退组和消退对照组的不僵立时间百分比在恐惧建立后第2天和第4天均显著低于正常组(86.03±8.90,P <0.01);到恐惧建立后第8天,消退组基本恢复正常,而消退对照组(36.18±12.60)仍低于正常组(P <0.01)。同组内不同时间点的比较:随时间进展,两实验组的恐惧水平均呈逐渐下降的趋势(P <0.01)。
(2)免疫组化结果显示:在恐惧建立后第2天,消退组大鼠IL区Cdk5的免疫反应阳性细胞数与正常组无差异,消退对照组的阳性细胞数(429±70)却多于正常组(322±52,P =0.026)和消退组(358±63,P <0.05);在恐惧建立后第4天,消退组的阳性细胞数明显减少(208±30,P <0.01),消退对照组基本恢复正常;在恐惧建立后第8天,消退组也恢复正常,与消退对照组无统计学差异。
(3)Western blot的结果为:在消退训练后1周内,消退组大鼠IL区Cdk5的蛋白表达呈减少的趋势:消退后第1和3天的表达与正常组的差异无统计学意义,而到消退后第7天(1.35±0.13)少于正常组(1.89±0.06,P <0.05);消退对照组的Cdk5蛋白表达在恐惧建立后第4天增多(2.94±0.19),明显高于消退组(1.78±0.20,P <0.01)和正常组(P <0.01);到恐惧建立后第8天基本恢复正常,但显著多于消退组(P <0.01)。
两组P35的表达与正常组无显著性差异;而两组P25的蛋白表达均显著高于正常组。在消退训练后1周,消退组P25的表达逐渐减少:消退后第7天(1.60±0.12)明显低于消退后第1天(2.14±0.24,P =0.032)和第3天(2.17±0.21,P =0.026);消退对照组在这个过程中则有增加的趋势:恐惧建立后第2天的表达(1.78±0.16)明显少于恐惧建立后第4天(2.50±0.19,P =0.039)和第8天(2.58±0.40,P =0.027)。
(4)液闪法的结果为:消退组大鼠IL区Cdk5的活性在消退训练后第1和3天显著增加(产物的放射性活度值分别为3107.78±536.42和3330.05±597.82,P <0.01),但到消退后第7天(2179.89±327.20)基本恢复到正常水平(1812.08±175.21);而消退对照组在这三天均明显高于正常组(P <0.05或P <0.01)。两实验组比较显示,在恐惧建立后第2天无统计学差异,消退组的激酶活性在恐惧建立后第4和第8天都显著低于消退对照组(P <0.05或P <0.01)。
结论:条件性恐惧使IL区Cdk5的蛋白表达和活性升高,大鼠的僵立行为较多;而经过消退训练后,Cdk5的蛋白表达逐渐降低、活性较快恢复正常,大鼠的恐惧反应也逐渐减少,这些变化可能有利于消退记忆的保持。
实验二、条件性恐惧消退训练后大鼠边缘下区突触数密度及突触界面结构的变化
目的:探讨条件性恐惧消退训练后,大鼠IL区突触数密度和突触界面结构的变化。
方法:将雄性SD大鼠随机分为消退组、消退对照组和正常对照组。采用声音结合足底电击的方式建立条件性恐惧模型,消退组在恐惧建立后24h进行消退训练,消退对照组不进行消退训练,两组均在不同时间点(恐惧建立后第8和22天)测定大鼠的僵立行为,然后快速断头取IL区按常规方法制作透射电镜标本,并用透射电镜观察、摄像,分析该区突触数密度和突触界面结构参数,包括:突触界面曲率、活性区长度、突触间隙宽度和突触后致密物质(postsynaptic density,PSD)厚度的变化。
结果:(1)行为学:消退组大鼠在两个时间点的保持成绩均与正常组(91.17±3.07)无显著差异;消退对照组在恐惧建立后第22天(66.73±8.75)的不僵立时间多于第8天(38.40±10.12,P =0.021),却都明显少于正常组(P <0.01)和消退组(P <0.05或P <0.01)。
(2)突触数密度:在两个时间点,消退组大鼠IL区的突触数密度(单位体积下的突触数量)均明显高于正常组(0.36±0.06,P <0.05);而消退对照组在恐惧建立后第8天的突触数密度(0.39±0.04)与正常无差异,但在恐惧建立后第22天(0.22±0.03)明显低于正常组(P =0.038)和消退组(0.48±0.06,P <0.01)。
(3)突触界面参数:三组间比较显示,突触界面曲率和突触间隙无统计学差异,而活性区长度和PSD厚度有显著性差异。与正常组相比,在消退训练后第7天,消退组大鼠IL区的PSD厚度无明显变化,到消退后第21天,PSD厚度增加(53.87±11.39,P =0.017);消退对照组则在两个时间点均表现为PSD的厚度变薄(P <0.05或P <0.01),且突触活性区长度均小于消退组(P <0.05或P <0.01)。
结论:条件性恐惧的建立使IL区的突触数密度减少,突触的结构受到损害,而消退训练后,该区的突触数密度增加,突触传递效率增强,有利于条件性恐惧反应的减少。
总之,从这两个实验可以推测:条件性恐惧消退训练后,IL区Cdk5的改变与IL区突触结构可塑性的变化存在一定的联系,即条件性恐惧建立后,大鼠IL区的Cdk5活性和P25表达持续升高,可能通过减少突触密度、改变突触结构,使该区的突触功能下降,大鼠的恐惧反应持续存在;然而,消退训练则使P25的表达逐渐下降、Cdk5活性较快恢复正常,可能有利于突触的结构可塑性,在行为上表现为大鼠的僵立时间百分比能较快恢复正常水平。
In recent years, catastrophic events occur so frequently that the incidence of post-traumatic stress disorder (PTSD) has a gradual growth. whereas, the pathogenesis of PTSD remains elusive, which leads to many difficulties to cure this disease. Recently, more and more scientists concern about PTSD. At present, the exposure therapy is usually used in treatment of PTSD, that is fear extinction, but fear memory easily appears again. This suggests that retention disorder of extinction memory may be a key factor in the occurrence and treatment of PTSD. Previous studies showed that the functional status of prefrontal cortex (PFC) was closely related to the occurrence and development of PTSD, and infralimbic cortex (IL) of PFC in rodents was an important site to store the extinction memory. However, it is not clear which substances involve in the process of retention of extinction memory in IL.
The serine/threonine kinase, cyclin-dependent kinase 5 (Cdk5) and its activator P35, which widespread in the cerebral cortex, could mediate functional and structural plasticity of synapes, and also affect the formation of fear memory and the acquisition of extinction memory. After extinction training, how do the Cdk5 expression and kinase activity change in IL of rats, and what changes will be observed about the synaptic structure in IL area. The present research aimed to answer these questions by two experiments.
The first experiment
Changes of Cdk5 expression and kinase activity in infra-limbic cortex of rats after conditioned fear extinction training
Objective: To investigate the changes of Cdk5 expression and kinase activity in IL area of rats after extinction training.
Methods: Male adult SD rats were randomly divided into extinction group (receiving fear conditioning and extinction training, EXT group), conditioned fear group (receiving fear training but no extinction training,Cont group) and natural comparison group(without any disposal, Naive group). Conditioned fear model of rats was established by six tones (unconditioned stimulus) paired with foot electric shock (conditioned stimulus). The extinction trials, which were given to animals at 24 hours after the last conditioning trail, were 16 tones without electric shock. Freezing behavior, expression and activity of Cdk5 were assessed on different days (2d, 4d and 8d) after fear conditioning.
Results:
(1) The freezing behaviors of EXT group and Cont group were more than naive group (86.03±8.90) on 2d and 4d (P <0.01). Until the 8d after fear conditioning, the freezing score of EXT group declined to the level of naive group, but it was less than that in Cont group (36.18±12.60, P <0.01).
(2) On 2d, the number of immunoreactive positive cells of Cdk5 significantly increased in IL area of Cont group (429±70, P <0.05). On 4d and 8d, there were no disparities between Cont group and naive group. Whereas the number of cells in EXT group decreased on 4d (208±30, P <0.01), but returned to normal on 8d.
(3) Western blot results showed: compared with naive group (1.89±0.06), the expression of Cdk5 in IL area of EXT group had a tendency to decrease, and the expression was lowest on 8d (1.35±0.13, P <0.05). While the expression of Cdk5 in Cont group increased on 4d (2.94±0.19), which was more than that in naive group and EXT group (1.78±0.20, P <0.01), and recovered almost to normal on 8d. No significant differences were found in the expression of P35 among the three groups, but the expression of P25 in experiment groups all were higher than that in naive group. After fear conditioning, the expression of P25 gradully increased, and reached peak on 8d (2.58±0.40,P <0.05). After extinction trials, the expression of P25 gradully decreased, and was lowest on 8d (1.60±0.12,P <0.05).
(4) After fear conditioning, the activity of Cdk5 enhanced. The activities in Cont group were higher than that in naive group (1812.08±175.21, P <0.05 or P <0.01) on 2d, 4d, and 8d. In contrast, although the activities of Cdk5 in EXT group had an obvious increasing on 2d (3107.78±536.42, P <0.01) and 4d (3330.05±597.82, P <0.01), the activity retured to normal level again(2179.89±327.20) on 8d, but was lower than that in Cont group (P <0.01).
Conclusion: After fear conditioning, the expression and activity of Cdk5 in IL area may increase, and the freezing behaviors of rats continue for a long time. But extinction training diminishes its expression and activity, which may be good for the retention of extinction memory, so that the fear response return to normal quickly.
The second experiment
Changes of synaptic numerical density and texture parameters analyses in infra-limbic cortex of rats after conditioned fear extinction training
Objective: To observe the changes of synaptic numerical density and texture parameters in IL area of rats after extinction training.
Methods: Male adult SD rats were randomly divided into EXT group, Cont group and Naive group (natural comparison group). EXT group and Cont group were all given 6 tone-foot shock paired trainings. But extinction trials were only given to EXT group 24 hours after fear conditioning. Behavioral tests were done at different days (8d and 22d) after fear training, and transmission electron microscope was used to observe the synaptic numerical density and texture parameters, including the thickness of postsynaptic density (PSD), the width of synaptic cleft, the curvature of synaptic interface and the length of active zone.
Results:
(1) No statistical differences were observed in freezing behavior between the EXT group and naive group on 8d and 22d. The freezing scores of Cont group had a trend to decrease, but the scores were all higher than that in EXT group and naive group on 8d and 22d (P <0.05 or P <0.01).
(2) The synaptic numerical densities in IL area of EXT group all were more than that in naive group (0.36±0.06, P <0.05) on 8d and 22d. Whereas on 8d after fear conditioning, the synaptic numerical densities in Cont group was equal to that in naive group, and decreased on 22d (0.22±0.03, P <0.05).
(3) The results of texture parameters analysing showed: there were no statistical differences in the curvature of synaptic interface and the width of synaptic cleft among the three groups. Compared with naive group, the thickness of PSD and the length of active zone had not obvious changes in EXT group on 8d, but the PSD became thickening notably on 22d (53.87±11.39, P <0.05). For the Cont group, the thickness of PSD was thinner than that in naive group and EXT group (P <0.05 or P <0.01) on 8d and 22d, and the length of active zone was less than that in EXT group (P <0.05 or P <0.01).
Conclusion: The establishment of the conditioned fear may decrease the synaptic numerical density in IL area of rats, and impair the synaptic structure. However, after extinction training, the synaptic numerical densities increase, and synaptic function could be enhanced, these changes may be good for fear extinction.
From these two experiments, it is possible that there is a relationship between Cdk5 and the changes of synaptic structural plasicity in IL area of rats. After fear conditioning, hyperexpression of P25 sustained more than one week, which caused Cdk5 activity increasing continuely, and could impair the synaptic structural plasticity of IL area. These changes may result in freezing behavior of rats. Through extinction training, the expression of P25 decreased gradually, and the kinase activity retured to normal fastily, which may be beneficial for the synaptic structural plasticity and conditioned fear extinction.
引文
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[5] Hawasli HA, Benavides RD, Nguyen C, et al. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation. Nat Neurosci, 2007, 10(7): 880-886.
[6] Hawasli AH, Bibb JA. Alternative roles for Cdk5 in learning and synaptic plasticity. Biotechnol J, 2007, 2(8): 941-948.
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[10] Schuman EM, Murase S. Cadherins and synaptic plasticity: activity-dependent cyclin-dependent kinase 5 regulation of synaptic beta–catenin–cadherin interactions. Biol Sci, 2003, 358(1432): 749-756.
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[2] Arnsten AF. Stress signalling pathways that impair prefrontal cortex structure and function. Nat Rev Neurosci, 2009, 10(6): 410-422.
[3] Holmes A and Wellman CL. Stress-induced prefrontal reorganization and executive dysfunction in rodents. Neurosci Biobehav Rev, 2009, 33(6): 773-783.
[4] Milad MR, Rauch SL, Pitman RK, et al. Fear extinction in rats: implications for human brain imaging and anxiety disorders. Biol Psychol, 2006, 73(1): 61–71.
[5] Luethi M, Meier B, Sandi C. Stress effects on working memory, explicit memory and implicit memory for neutral and emotional stimuli in healthy men. Front Behav Neurosci, 2009, 2(5): 1-9.
[6] Alexander JK, Hillier A, Smith RM, et al. Beta-adrenergic modulation of cognitive flexibility during stress. J Cogn Neurosci, 2007, 19(3): 468-478.
[7] Shin LM, halen PJ, Pitman PK, et al. An fMRI study of anterior cingulate function in posttraumatic stress disorder. BiolPsychiatry, 2001, 50(12): 932-942.
[8] Quirk GJ and Sotres-Bayon F. Prefrontal control of fear: more than just extinction. Current Opinion in Neurobiology, 2010, 20(2): 231-235.
[9] Quirk GJ, Russo GK, Barron JL, et al.The role of ventromedial prefrontal cortex in the recovery of extinguished fear. J Neurosci, 2000, 20(16): 6225–6231.
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