Study On The Mechanisms Of Reactivation Of Fear Memory And The Protective Effects Of Curculigoside

Background Learning & memory are inherent abilities for the individuals to survive and acquire intelligence.Learning and memory are comprised of a series of different processes,including information acquisition,consolidation,reactivation,reconsolidation,or extinction,etc.Fear memory is one type of memory,which includes all processes of memory and performs important roles in dealing with complex environments.However,fear memory can also participate in the regulation of human emotional changes.Studies have shown that "reactivation" or "repeated activation" of fear memory can lead to depression-like and anxiety-like behaviors,which threaten human life quality.Thus,to reveal the mechanism of the "reactivation" of fear memory will be of great significance to find treatment for anxiety-like and depression-like behaviors.Calpain is a class of important calcium-dependent proteases,which function through degrading its substrates.Previous results have shown that calpain has critical action in memory acquisition and consolidation.However,the function of calpain performed in memory reactivation is still unknown.Curculigoside(CUR)is one of the main active ingredients of rhizome,a perennial herb of red spider lily(Curculigo orchioides Gaertn).Modern pharmacological research shows that CUR has a wide range of pharmacological activities,such as anti-oxidation,protection of cardiovascular disease,osteoporosis,enhancement of immune function and neuroprotection.In addition,it has been reported that CUR possesses the ability to ameliorate depression and anxiety-like activities.The present study was conducted to further investigate its effect on fear memory,in order to provide a reference for the mechanism of anti-depression and anxiety.1 Objective This study aimed to investigate the effects of calpain inhibitor-calpeptin on fear memory "reactivation",and to assess their mechanisms;to evaluate the effect of CUR on freezing in fear conditioning task.2 Methods Behaviors: A conditioned stimulus-unconditioned stimulus(CS-US)model was applied to study the memory consolidation,reconsolidation and extinction.In each type of model,calpeptin(2 mg/kg,i.p.)was intraperitoneally injected 30 min before or immediately after electrical stimulation,re-exposure.The experiments were divided into a calpeptin group and a control group.The mice in control group received a similar volume of saline.The freezing was applied to represent the memory.Molecular mechanisms: The potential substrates of calpain were selected to disclose the mechanisms.Immediately after re-exposure,amygdala was isolated.The levels of PP2A-B56-?,SCOP,PTEN,p-CREB,p-m Tor and other kinases were detected after re-exposure by western blot and immunofluorescence staining.Intervention of curculigoside: curculigoside(20 mg/kg/d,i.p.,7 days)was administered before CS-US(before adaption)or after CS-US(after re-exposure).A control group was also set in the experiment.The effects of curculigoside on fear memory were investigated preliminarily by detecting the change of freezing.3 Results Effects and mechanisms of calpeptin on fear memory "reactivation" and underlying mechanism:In the memory experiment,pre-electrical stimulation application of calpeptin significantly decreased the freezing time compared with control group(P<0.05).By contrast,post-electrical stimulation application of calpeptin did not affect the freezing time compared with control group(P>0.05).In the reconsolidation experiment,pre-re-exposure administration of calpeptin significantly decreased the freezing time compared with control group(P<0.05).By contrast,post-re-exposure administration of calpeptin did not affect the freezing time compared with control group(P>0.05).In the extinction experiment,pre-re-exposure administration of calpeptin significantly increased the freezing time compared with control group(P<0.05).Moreover,post-re-exposure administration of calpeptin also significantly increased the freezing time compared with control group(P<0.05).Western blot showed that the expression of PP2A-B56-?,p-m Tor and p-CREB in amygdala was significantly increased after 15 min re-exposure(P<0.05).By contrast,there was no significant difference regarding PP2A-B56-?,p-m Tor and p-CREB expression between pre-and post-exposure in calpeptin group(P> 0.05).Immunofluorescence staining showed that p-m Tor and PP2A-B56-? were mainly expressed in the cytoplasm of amygdala neurons,while p-CREB was mainly located in the nucleus.In control group,the numbers of PP2A-B56-?,p-m Tor and p-CREB positive cells in the amygdala were significantly increased after the 15 min re-exposure.By contrast,there was no significant difference in the number of PP2A-B56-?,p-m Tor and p-CREB-positive cells before and after re-exposure in calpeptin group.Additionally,the expression levels of SCOP,PTEN,p-Akt,Akt,p-Erk and Erk protein in the amygdala after re-exposure were not significantly altered compared with pre-re-exposure(P> 0.05).The influence of curculigoside on "reactivation" of fear memory:Pre-CS-US administration of curculigoside did not affect the freezing time compared with control group(P>0.05).By contrast,post-CS-US administration of curculigoside significantly increased the freezing time compared with control group(P<0.05).4 Conclusion Calpain participates in the "reactivation" process of fear memory.Pre-re-exposure inhibition of calpain activity may inhibit memory reconsolidation or extinction induced by "reactivation".The mechanisms may be related to the synthesis of PP2A-B56-?protein mediated by m Tor pathway.Curculigoside like has certain effect on fear memory,and it may inhibit memory extinction after the formation of contexual fear memory.