Studies On The Effect And Mechanism Of The Extinction Retention Of Conditioned Fear Memory In The Infralimbic Area Of Rat

With the accidental disaster on a rise, the post traumatic stress disorder (PTSD) has become a worldwide focus. Actually, PTSD is a series of disorders caused by traumatic-related memories (conditioned fear). The disorder is difficult to vanish naturally once it comes into being, and seriously affects people's daily life. The exposure therapeutics is based on erasing the conditioned fear and can not maintain the effect in treating PTSD, because the extinct fear recurs in a short time, suggesting that extinction retention disorder may be the key factor to cause PTSD and the difficulty in its consequent therapy. Previous experiments have shown that infralimbic (IL) in medical prefrontal cortex (mPFC) is closely interrelated to the morbility, development and therapy of PTSD, but its mechanism of retention of conditioned fear extinction is not clear.Based on the previous studies, in the current study, the best'time window'of conditioned fear memory extinction was investigated using behavioral pharmacology, morphological and Western Blot methods to illuminate the IL area mechanism of the retention of conditioned fear memory extinction. experimental proof to improve and consolidate PTSD therapy effect, and researching. To achieve this purpose, the following three experiments were performed. The first experiment Dynamic Observation on the extinction retention after operations with different extinction protocolsObjective: To investigate the dynamic change of memory retention of conditioned fear extinction after different extinction protocols.Methods: The rats'conditioned fear model was established with conditioned stimulus (classical auditory fear conditioning) pairing with unconditioned stimulus (an aversive foot shock). Later the repeated single conditioned stimulus was used for extinction training. 48 male SD rats were randomly divided into three groups: control group (natural extinction, Cont) in which rats were subjected to auditory conditioned fear but no fear extinction conditioning; immediate extinction group (Imme EXT) in which fear extinction started 15min after establishment of conditioned fear; delayed extinction group (EXT) in which fear extinction started 24h after establishment of conditioned fear. The freezing behavior was determined on half of rats in the three groups on 1st, 3rd, 7th, and 20th day after fear extinction. And the anxiety behavior was determined by elevated plus maze (EMP) method on the other rats before experiment and on 1st, 3rd, 7th, and 20th day after fear extinction.Results (1) There were no significant differences in baseline freezing levels among three groups (Cont, Imme EXT, EXT) (P>0.05). Fear extinction caused a significant stable and long-lasting extinction effect in EXT group compared to that in immediate extinction group and control group (P<0.001). Freezing behavior was decreased significantly from 1st to 3rd day after fear extinction in EXT group(P<0.01). Between Imme EXT group and Cont group was no significant difference(P>0.05).There was no significant difference in freezing behavior among the three groups from 7th to 20th day after fear extinction.(2) There was no statistically significant difference in baseline levels of The percentage of open arm entries (OA entries % )and The percentage of the time spent in the open arm entries( OA time % )among three groups (P>0.05). There was no significant difference between OA entries % and OA time% at different time period after fear extinction (P>0.05). Generally, the anxiety behavior level was decreased with the experiment time lasting, but there was no significant difference at the same time point among 3 groups (P>0.05). Conclusion The different extinction protocols cause different long-lasting extinction effects with the best effect in the delayed extinction group. Besides, with the similar anxiety behavior level among 3 groups, it is reasonable to rule out the possibility that the increase in anxiety level causes high level of freezing behavior in Immi EXT group.The second experiment Dynamic Change of pCREB in IL during retention of conditioned fear memory extinctionObjective: To investigate the change of extinction retention and expression of pCREB in rat's infralimbic area (IL) in different retention period after different extinction protocols.Methods: Experiment 1 showed that delayed extinction of conditioned fear had the significant effect compared with immediate extinction and the effect was obvious during 1st-7th day after fear extinction. The experiment 2 was to find out the dynamic change of pCREB in IL on 1st, 3rd, 7th day after delayed fear extinction for those rats subjected to auditory-foot shock pairing stimulus and unpaired stimulus. Male adult SD rats were randomly divided into 5 groups: natural comparison group (Na?ve, without any disposal), shame A group (Sham A, unpaired auditory and foot shock stimulus), shame B group (Sham B, only by foot shock stimulus), conditioned fear group (natural extinction, Cont)and delayed extinction group (EXT). Except in the conditioned fear group, the delayed extinction method in experiment 1 was employed in the other groups. Twenty-four hours after extinction, samples were collected in each group. Samples were collected in EXT group at different time point after delayed fear extinction (1st, 3rd and 7th day). Immunoflourence staining and Western Blot methods were conducted to locate neurons and determine the expression of pCREB in IL (3-4 rats/time point). The effect of conditioned fear on the expression of pCREB in IL was much more significant than that of unconditioned fear by using single Immunoflourence signal of pCREB in IL. In the triple immunoflourence signal, pCREB indicated cell nucleus (green), Neu-N perikaryons and nucleus (red) and Hoechst nucleus (blue). Cells with pCREB-positive, Neu-N-positive and Hoechst-positive staining were counted and recorded to show the expression of pCREB in IL neurons.Results (1) The percentage of freezing time in EXT group 1d (48.72±10.27)after extinction was significantly lower than with those in Sham A (79.65±6.88), Sham B (77.53±6.03) and Cont group (61.12±10.22) respectively, while it was significantly higher than that in Na?ve group (55.77±10.40)( P<0.01). There was no significant difference among Shame A, Shame B and Cont group(P >0.05).The count of pCREB-positive cells in IL in EXT group 1d (823.70±67.31)after extinction was significantly higher than that in Sham A(442.20±53.19), Sham B (453.20±49.47) and Cont group (564.20±55.37) respectively(P<0.001), while it was significantly lower than that in Na?ve group (994.00±56.62)(P<0.01).(2) The expression of pCREB-positive neurons in IL with triple immunofluerence staining: The expression of pCREB-positive neurons in IL was gradually increased on 1d (45±5.70), 3d (62±8.39) and 7d (66.2±7.59) after delayed extinction, of which the expression was increased dramatically from 1d to 3d (P<0.001), while there was no significant difference between the expressions from 3d to 7d (P>0.05). Seven days after the extinction, the expression recovered almost to the same level as that in Na?ve. The expression of pCREB-positive neurons in IL in conditioned fear group (28±2.73) was significantly lower than that of na?ve(73.2±5.31) and delayed extinction group at different time points (P<0.001).(3) Western Blot results showed: Gray scale of pCREB protein toβ-actin showed gradual increase from 1st (0.86±0.06), 3rd (1.11±0.06) to 7th (1.22±0.04) day in delayed extinction group(P<0.01), of which it was increased dramatically from 1d to 3d. The expression of pCREB was significantly decreased in conditioned fear group (0.54±0.03) (P<0.001), and recovered to the expression level in na?ve(1.39±0.05) 7d after extinction (P>0.05).Conclusion: The auditory fear conditioning can cause the reduced expression of pCREB in IL, while the improved freezing behavior can lead to the increased expression. The expression of pCREB in IL was significantly increased 1-3d after the extinction in IL neurons , suggesting that neurons may be implicated in the extinction retention.The third experiment NMDA receptor's function in retention of conditioned fear extinctionObjective: To observe the effects of MK-801, noncompetitive NMDA receptor antagonist, on the conditioned fear extinction retention and the expression of pCREB in IL for investigating the possible mechanism.Methods: Delayed extinction method was employed on male adult SD rats which were randomly divided into 4 groups. Pre-MK-801 group: Intraperitoneal injection of MK-801(0.3mg/kg) was given to rats 40 min before extinction training; Post-MK-801 group: Intraperitoneal injection of MK-801(0.3mg/kg) was given to rats 4 h after extinction training; EXT group: Extinction training was conducted 24 h after conditioned fear memory was established and intraperitoneal injection of equal amount of saline was given to rats; Cont group: The conditioned fear memory was established without any extinction training, and intraperitoneal injection of equal amount of saline was given to rats. The freezing behavior time and retention study of fear extinction were performed with half quantity of the rats separately before experiment, on the day of extinction (EXT-0d), and 1st, 3rd, 7th day after extinction (EXT-1, 3, 7d). The rats were executed to death for immunohistochemistry and western blot analysis (3-4 rats/time). In the triple immunoflourence signal, pCREB indicated cell nucleus (green), Neu-N perikaryons and nucleus (red) and Hoechst nucleus (blue). Cells with pCREB-positive, Neu-N-positive and Hoechst-positive staining were counted and recorded to show the expression of pCREB in IL neurons.Results (1) There were no significant differences in freezing behavior among four groups (Pre-MK-801, Post-MK-801, EXT, Cont) (P>0.05). Freezing behavior on EXT-7d was significantly lower than that on EXT-0d in Cont group. There were no significant differences in freezing behavior between Pre-MK-801 group and Cont group on the 1st, 3rd and 7th day after fear extinction(P>0.05). Freezing behavior in Post-MK-801 group was significantly higher than that in EXT group, but lower than that in Pre-MK-801 group and Cont group on the 1st, 3rd and 7th day after fear extinction (P<0.05). There were no significant differences in freezing behavior between Pre-MK-801 group and Post-MK-801 group at different time points after fear extinction.(2) There was no statistically significant difference in baseline levels of the percentage of open arm entries (OA entries % )and the percentage of the time spent in the open arm entries( OA time % ) among four groups (P>0.05). There was no significant difference between OA entries % and OA time% at different time period after fear extinction (P>0.05). Generally, the anxiety behavior level was decreased with the experiment time lasting, but there was no significant difference at the same time point among 4 groups (P >0.05).(3) The expression of pCREB-positive neurons in IL with triple immunofluerence staining: The expression of pCREB-positive neurons in IL in Post-MK-801 group (42.80±4.32) was lower than that in EXT group(48.80±2.39) (P<0.05), but significantly higher than that in Pre-MK-801 group (34.80±3.11) and Cont group (35.60±4.16) (P<0.05), There was no significant difference between Pre-MK-801 group and Cont group of the expression of pCREB-positive neurons in IL (P>0.05). (4) Western Blot results showed: Gray scale of pCREB protein toβ-actin showed that the expression of pCREB protein in Post-MK-801 group (0.71±0.06) was significantly lower than that in EXT group(0.87±0.08) (P<0.01), but significantly higher than that in Pre-MK-801 group (0.49±0.05)and Cont group(0.52±0.03) (P<0.01). There was no significant difference between Pre-MK-801 group and Cont group (P>0.05).Conclusion Intraperitoneal injection of MK-801(0.3mg/kg) 40 min before extinction training or 4h after extinction training was given to rats that has no influence on the change of anxiety behavior of rats but affects their extinction retention of conditioned fear. The way of administration causes thedifferent change of the expression of pCREB in IL, which is limited to the neuron. The effect of extinction in Pre-MK-801 is worse than that in Post-MK-801. The expression of pCREB in IL may participate in the extinction of conditional fear memory. MK-801 may antagonize NMDA receptor to restrain the extinction of conditional fear memory.