The Relationship Between Hippocampal Racl Activity And The Maintenance And Forgetting Of Contextual Fear Memory

Objective:To investigate the role of hippocampal Rac1activity in the maintenance and forgetting of contextual fear memory, explore the molecular and cellular mechanisms underlying hypermnesia of the trauma in the patients suffering posttraumatic stress disorder (PTSD) and provide a new theoretical guide for the treatment of PTSD.Methods:Experiment1:To ptimized a protocol that induced a similar learning curve but resulted in different contextual fear memory. Adult male Sprague-Dawley rats received5trials of electrical footshocks at an intensity of either0.5,0.8or1.2mA for1s. The5trials were presented at inter-trial intervals (ITI)12s (massed),122s (spaced) or600s (long spaced).24h later, rats were placed into the conditioned chamber to test for contextual fear memory.Experiment2:To investigate the relationship between contextual fear memory and the level of Racl activity in the hippocampus in rats. Adult male Sprague-Dawley rats received5trials of electrical footshocks at an intensity of0.8mA for1s. The5trials were presented at ITI12s,122s or600s.1h,24h or7d later, rats were placed into the conditioned chamber to test for contextual fear memory.Some of the rats were sacrificed at0h,1h or24h after fear conditioning for detecting the activity of Rac1by using western blotting and immunohisochemistry.Experiment3To investigate whether Racl activity in the hippocampus regulates contextual fear memory in rats. Adult male Sprague-Dawley rats received5trials of electrical footshocks at an intensity of0.8mA for1s. The ITI of the5trials were presented at12s or122s. Immediately after training for contextual fear conditioning, the Racl inhibitor NSC23766was injected into the bilateral CAl areas of the hippocampus in12s group, while the Racl activator CN04-A was administrated to the bilateral CAl areas of the hippocampus in122s group before contextual fear conditioning.24h and (or)7d later, rats were placed into the conditioned chamber to test for contextual fear memory.Experiment4To investigate the relationship between contextual fear extinction and levels of Rac1activity in the hippocampus in rats. Adult male Sprague-Dawley rats received5trials of electrical footshocks at an intensity of1.0mA for1s. The ITI of the5trials were122s.24h later, Rats received6trials of extinction training, each extinction training lasting5min. The ITI of the6extinction trials were0min,10min or30min.24h and16d later, rats were placed into the conditioned chamber test for the fear memory. Some of rats were sacrificed at1h after fear extinction for detecting the activity of Rac1by using western blotting and immunohisochemistry.Experiment5To investigate whether Rac1activity in the hippocampus directly regulates the extinction of contextual fear memory in rats. Adult male Sprague-Dawley rats received5trials of electrical footshocks at an intensity of 1.0mA for1s. The ITI of the5trials were122s.24h later, Rats received6trials of extinction training, each extinction training lasting5min. The ITI of the6extinction trials were0min. Immediately after last extinction training, the Rac1inhibitor NSC23766was injected into the bilateral CAl areas of the hippocampus.24h and16d later, rats were placed into the conditioned chamber to test for extinction memory.Experiment6To further investigate whether Rac1activity in the hippocampus directly regulates the extinction of contextual fear memory in rats. Adult male Sprague-Dawley rats received5trials of electrical footshocks at an intensity of1.0mA for1s. The ITI of the5trials were122s.24h later, Rats received6trials of extinction training, each extinction training lasting10min. The ITI of the6extinction trials were0min,10min or30min.24h and16d later, rats were placed into the conditioned chamber test for the fear memory. Some of rats were sacrificed at1h after fear extinction for detecting the activity of Racl by using western blotting and immunohisochemistry. Results:Experiment1At an intensity of0.5mA, three group animals show similar contextual fear memory when examined24h after training. At an intensity of0.8and1.2mA, the contextual fear memory significantly heightened in the spaced but not massed training groups. The results show that the electrical footshocks at0.8mA was optimal for our objective.Experiment2Animals in spaced group (122s) presented a significantly heightened contextual fear examined at1h,24h and7d after training; the long-spaced group (600s) showed a relatively lower heightened contextual fear at24h and7d after training and a relative higher heightened contextual fear at1h compared to the spaced group; the massed group (12s) showed the lowest level of contextual fear at1h,24h and7d after training. The western blotting results show that compared with naive controls, Rac1activity in the hippocampus was no difference among the three groups at0h after contextual fear conditioning, but largely inhibited in the spaced groups (122s and600s), and the lowest level of Racl activity was found in the122s group1h after contextual fear conditioning, and the inhibition alleviated24h after contextual fear conditioning. In contrast, compare to naive controls, Rac1activity in massed group (12s) remained unchanged1h and24h after contextual fear conditioning. The immunofluorescence (IF) results show that the staining of Racl-GTP in the CAl area of hippocampus in spaced but not massed training animals were sharply weakened compared to naive rats1h after contextual fear conditioning. The results show that spaced training inhibited Rac1activity in the hippocampus and caused hypermnesia of contextual fear.Experiment3The contextual fear memory examined at24h and re-examined at7d after the massed training (12s) was largely heightened by the Rac1inhibitor similar to the heightened memory in the spaced training group. Contextual fear memory directly examined at7d after training was also heightened by the Rac1inhibitor. On the contrary, Compared with vehicle control aminals, the Racl activator CN04-A reduced contextual fear memory in the spaced training group (122s) when examined at24h and re-examined at7d after training. The results show that regulation of Rac1activity in the hippocampus regulated contextual fear memory in rats.Experiment4The result of30min extinction training show that the extinction curve in the massed extinctin group was dramatically decreased, whereas in the spaced groups only showed a less decreased extinction curve. When examined24h after extinction training the contextual fear memory among the three groups was not different, but when reexamined16d after extinction training, it was spontaneously partially recovered in the spaced group and nearly fully recovered in the long-spaced group. The western blotting results show that the levels of active Racl-GTP relative to total Racl were robustly enhanced in the massed group when compared with the naive or spaced groups, and Racl activity in the hippocampus was slightly inhibited in the30min group. The immunofluorescence (IF) results show that the staining of Racl-GTP in the CAl area of hippocampus in massed extinction training animals were robust enhanced while in30min group were slightly weakened compared to naive rats. The results show that massed extinction training promoted Rac1activity in the hippocampus and caused obvious extinction of contextual fear.Experiment5A slightly heightened contextual fear memory was found in the Racl inhibitor-treated group compared with vehicle control when examined at24h after extinction, and the heightened contextual fear memory became prominent when re-examined at16d after extinction. The contextual fear memory was nearly fully recovered in the Racl inhibitor-treated group while nearly completely suppressed in vehicle control sample. The results show that inhibition of Rac1activity in the hippocampus prevented the extinction of contextual fear induced by massed extinction training.Experiment6The result of strong extinction training show that the contextual fear memory in all animal groups was similarly decayed at24h and16d after extinction. The western blotting results show that the level of active Rac1-GTP relative to total Racl was enhanced in massed and spaced extinction group, and not inhibited in long spaced extinction group, whereas a significantly higher level of Racl activity was found only in the massed training group. The immunofluorescence (IF) results show that compared with naive controls, the staining of Racl-GTP in the CA1area of hippocampus enhanced in massed and spaced extinction group, and no changed in long spaced group. The results show that promotion of recovery of the inhibited Rac1activity in the hippocampus in spaced extinction training rats facilitated extinction of contexual fear memory. Conclusion:Inhibition of Rac1activity in the rat hippocampus led to heightened contextual fear, promotion of Rac1activity led to weakened contextual fear. Rac activity regulated forgetting in drosophila, thus, we proposed that Racl activity regulates the forgetting of contextual fear conditioning in rats, and we suggested that inhibition of Racl activity likely caused hypermnesia of contextual fear and might contribute to the pathological fear of PTSD.