Studying The Role Of Microglia In Contextual Fear Memory Linking Both Under Physiological Condition And After PTSD-associated Stress Exposure

Memories formed in a particular context in the real world are not isolated.Time is an important variate in memory organization.The association of memories acquired at different time is an important part of our daily life.Evidence from humans and animal studies indicates that memories that occurred close in time are more likely to be meaningfully associated.When we retrieve one memory,other associated memories are also prone to be retrieved,whereas those with a longer experience interval are not.According to a recent study,CCR5 is a key molecular modulator to shape the time window of memory linking.Additionally,locus coeruleus-d Cornuammonis(CA1)-projecting neurons specifically regulate memory linking in a dopamine-dependent manner.Regardless of this,the molecular and cellular mechanisms that shape the temporal window for memory linking are largely unknown.Post-traumatic stress disorder(PTSD)is a mental illness that develops after a traumatic event,characterized by repeated recall of the traumatic event,avoidance,negative emotions and thoughts,and hyperarousal state.Plenty of studies have demonstrated that PTSD is manifested by multimode cognitive and memory deficits,such as over generalization of fear memory and impaired fear memory extinction.Traumatic stress can lead to abnormal memories,which in turn exacerbate emotional abnormalities in PTSD.Whether traumatic stress leads to abnormal memory linking and underlying neuro-immune mechanisms are key questions that we are interested to study.Microglia are the main immune cells resident in the central nervous system(CNS).In addition to initiate immune response,microglia are also considered as a key regulator of neuronal activity and synaptic plasticity,and thus participate in the process of learning and memory.The role of microglia in single memory process has been extensively studied.However,whether microglia play an important role in memory linking remains unanswered.Evidence from animal studies indicates that PTSD-associated stress induces abnormal activation of microglia,and depletion of microglia can improve PTSD-associated cognitive impairment.Clarifying the role of microglia in memory linking and PTSD-related memory linking deficits will help to understand the mechanisms of memory linking.More importantly,it will provide new insights and potential therapeutic targets for memory disorders associated with neuropsychiatry diseases,such as PTSD.The purposes of our study:1.To identify the time window for memory linking and the involvement of microglia in physiological memory linking.2.To check whether memory linking is abnormal in PTSD and to explore the role of microglia in this process.Methods:Single prolonged stress(SPS),a frequently used animal model of PTSD,was adopted in this study.The anxiety-related behaviors were assessed with elevated plus maze(EPM)tests.Modified contextual fear conditioning paradigm was used to evaluate time window of memory linking.Real-time PCR was used to quantify the relative expression of proinflammatory cytokines in the hippocampus.Immunostaining was used to measure the number and morphological changes of microglia.Finally,RAM virus injection and dox system were used to investigate the cellular mechanism of memory linking.Results:1.The time window for physiological memory linking is between 24 h and 3d.Two contextual memories occurred with intervals of 5h to 24 h can link together,while two memories with an interval of 3d cannot.Specifically,the freezing level during memory tests was significantly higher in 5h and 24 h linking contexts than that in the novel context,while there was no difference between 3d linking context and novel context.2.PLX5622 can effectively deplete microglia in the hippocampus.3.Microglia depletion by PLX5622 significantly extended the time window for contextual memory linking.Specifically,mice showed significantly higher freezing in the3 d linking context than that in the novel context.Microglia depletion did not affect shockcontext freezing during memory tests.4.SPS facilitated anxiety-like behaviors.In the EPM tests,stressed mice exhibited dramatic decrease in the percentage of exploration time spent in open arms.5.SPS did not affect baseline freezing in context.In details,stressed mice showed similar freezing as control non-stressed mice in all contexts before conditioning.6.SPS exposure did not affect the formation of single contextual fear memory.Specifically,stressed mice and control non-stressed mice showed similar freezing in shock context during memory tests.7.SPS exposure led to excessive memory linking for different context.Specifically,the freezing levels of stressed mice were significantly higher in both 5h and 3d linking context than in novel context during memory tests,suggesting contextual memory linking between different contexts.In contrast,non-stressed control mice showed memory linking only at time interval of 5h.8.SPS exposure prolongs the time window for overlapping between neuronal population encoding different contexts.9.SPS exposure significantly increased the number and changed morphology of microglia in the hippocampus,characterized by enlarged soma size,reduced branch length and arborization.In addition,SPS exposure also dramatically increased the transcripts of Interleukin-6(IL-6)and Tumor necrosis factor-α(TNF-α)in the hippocampus.10.Microglia depletion improved excessive linking for contextual memories induced by SPS exposure.The freezing level of PLX5622-treated SPS mice was significantly higher in the 5h linking context than in the novel context.In contrast,the same group of mice showed similar freezing in 3d linking context and novel context.The control SPS mice without microglia depletion showed higher freezing in both 5d and 3d linking contexts than in novel context.In summary,we found that the time window for contextual memory linking under physiological conditions was between 24 h and 3d.Depletion of microglia with PLX5622 extended the time window for memory linking to more than 3d,indicating that physiological microglia play a negative role in regulating memory linking process.However,SPS exposure enhanced overlapping between engram cells encoding different contextual memory and meanwhile facilitated linking between different contextual memories.SPS exposure significantly increased the number of microglia,and the transcripts of IL-6 and TNF-α in the hippocampus.In addition,microglia depletion with PLX5622 alleviates SPS-induced over-linking between contextual memories.Therefore,we speculate that SPS-induced pro-inflammatory activation of microglia in the hippocampus contributes to abnormal memory linking induced by SPS exposure.Altogether,our study provides first evidence that microglia regulate memory linking both under physiological condition and after PTSD-associated stress,providing important information for further exploring the neuro-immune mechanism of microglia in regulation of memory linking and PTSD-associated memory linking deficits.