Studies On The Changes Of Cdk5 And Synaptic Structural Plasticity Of Infra-limbic Cortex Of Rats After Conditioned Fear Extinction Training

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 experimentChanges of Cdk5 expression and kinase activity in infra-limbic cortex of rats after conditioned fear extinction trainingObjective: 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 experimentChanges of synaptic numerical density and texture parameters analyses in infra-limbic cortex of rats after conditioned fear extinction trainingObjective: 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.