The Study Of The Mechanism Of The Endocannabinoid System Involved In Regulating The Generalization Of Conditioned Fear Memory

Post-traumatic stress disorder(PTSD)is a kind of abnormal mental reaction which is caused by serious traumatic stress(such as war and natural disasters),it harms people's physical and mental health.With the frequent occurrence of accidents of war,social violence and natural disasters,the incidence of PTSD was also increased,but nowadays,there is not any effective means of treatment and prevention,for its pathogenesis is unclear.When PTSD patients see and hear the clues which are similar to the original traumatic experience,he/she may associate with original fear traumas,re-experience traumas,avoid things related to traumas,alert highly,feel frightening.Nightmares,insomnia,emotional numbness and personality change are also symptoms of PTSD.They could not distinguish present safe circumstances and original traumatic circumstances.The symptom is related with over-generalization of fear memory.Endogenous cannabinoids(eCB)are important bioactive substances,which are similar to natural cannabinoids in the body.It has been shown that there is a bidirectional regulation of fear and associated negative emotions such as anxiety,so eCB system is considered to be the buffer system of emotional response.Recently studies have shown that exogenous cannabinoids can inhibit the generalization of fear memory,suggesting that eCB system may be involved in the regulating of fear conditioning memory generalization.This study is based on the model of conditioning generalization of fear memory,and would explore the function of eCB in the process of generalization of fear memory by using pharmacological and genetic methods.In addition,using electrophysiological techniques,we want to clarify the neural mechanisms of fear conditioning memory generalization mediated by eCB system.(1)Establishing the modified model of fear memory generalizationWe modified the model based on the existing model of fear memory generalization;the percentage of "Freezing" in the generalization test was used as the standard of analysis.The accuracy of the model was investigated from three aspects:the interval time of electric stimulation,the intensity of footshock and the degree of environmental change.Finally,a simple,stable,reliable,effective and repeatable generalization model was established,which lay a solid foundation for the subsequent experiments.(2)The eCB system participates in the control of fear conditioning memory generalizationAfter intraperitoneal injection of CB1R antagonists AM281,freezing time in the generalization test showed a significantly higher percentage of the control,and discrimination index decreased significantly.It indicated that AM281 could reinforce the conditioning fear memory generalization in mice.After generalization test,the expression of CB1R had significant difference with control group,indicating the eCB system may be involved in the generalization of fear memory.The results of cfos phosphorylation proved that the hippocampus is one of the key brain regions during the generalization of fear memory.(3)The eCB system regulated fear memory generalization via GABA or glutamate pathwayWe explored low dose("ineffective")GABAA receptor agonist/antagonist and NR2A antagonist which had no effect on fear generalization behavior by microinjection of different doses of drugs,laying the foundation for further verify pathways of the eCB.Consistent with prior studies that AM281 could enhance the degree of generalization of fearful memory in mice by microinjection in hippocampus.This function can be facilitated/offset by preinjection subliminal GABAA receptor agonists/antagonists,and can also be reversed by the NVP under the sub-threshold.The results above indicated that the hippocampal eCB system was able to restrict the generalization of fear memory and may recruit GABA pathway and glutamate pathway.(4)The generalization of conditioning fear memory mediated by the eCB system depended on the activation of CB1R on GABAergic neuronsCB1R on GABAergic neurons specificity knockout mice exhibited an enhancement of fear memory generalization,while there was no significant difference between CB1R on glutamatergic neurons specificity knockout mice and the littermate.According to these results,we speculated that eCB system could regulate the generalization via activating of CB1R on GABAergic neurons,which was accompanied with the variations of related protein expressions.(5)Changes of synaptic plasticity associated with eCB system in fear memory generalizationUsing electrophysiological techniques,we observed high-frequency stimulation(HFS)evoked a sight LTP of fEPSPs in control mice after fear memory generalization test.In the CB1R deletion on GABAergic neurons(GABA-CB1R-/-)mice,LTP induction by HFS was failed,suggesting the eCB system can regulate the synaptic plasticity of excitatory neurons in CA1 area during the generalization of fear memory.In summary,the eCB system could restrict the generalization of fear memory,which possibly attributed to the disinhibition effect on GABAergic neurons via activating of CB1R on GABAerfic neurons,and then reducing the release of GABA neurotransmitters.The eCB system could also affect the synaptic plasticity of hippocampal excitatoryneurons indirectly to prevent overgeneralization.Therefore,this study provides a new approach to the pathogenesis of PTSD.A large amount of research data have been provided to clarify the fear memory generalization neural mechanism regulating by eCB system,which would contribute to find new treatments for mood disorders such as PTSD.