Microglia Mediate Forgetting Via Complement-dependent Synaptic Elimination

Learning and memory is one of the most important functions of the brain,and is the most famous research field.The hippocampus is an important brain region located between the thalamus and the medial temporal lobe,which is responsible for encoding and storing memories.Studies have shown that memory is coded and allocated to engrams cells within related brain regions.Synapses between engram cells are believed to be substrates for memory storage,while weakening or loss of these synapses leads to forgetting of related memories.Reactivation of engram cells is essential for memory recall,while failure in reactivation of engram cells leads to forgetting of related memory.Circuit rewiring and synaptic reorganization may lead to loss or weakening of synaptic connections between engram cells,thus resulting in forgetting of previously existing memories.Microglia,are an important type of glia in the brain,which account for about 10-15%of the total n?mber of brain cells.Previous studies have shown that microglia are the main immune cells in the central nervous system and play an important role in maintaining brain homeostasis.Recent studies have shown that microglia are not only important for pruning excessive synapses during postnatal brain development,but also involved in the dynamics of synapses even in the adult brain.Whether microglia cells can eliminate synapses in adulthood,and whether microglia cells can mediate memory forgetting,are questions that have not been systematically addressed.First,in our study,we used contextual fear conditioning?CFC?as a behavior paradigm.The mice could form contextual fear memory after 3 trainings,reflected by the freezing behavior in the training context.We observed decreased freezing of mice when tested 35 days post training?dpt?,compared to the freezing when tested 5 dpt.This suggests forgetting of contextual fear memory over time.We depleted microglia in C57BL/6 mice with PLX3397?PLX?,a CSF1R/c-kit antagonist,via mouse diet.PLX treatment significantly increased freezing of the animals,and reactivation rate of engram cells 35 days post training.This suggests that depletion of microglia prevents memory forgetting and engram dissociation.Next,we imaged microglia in the DG of adult CX3CR1^GFP/+ mice,in which microglia are GFP-labeled.When co-stained with synaptophysin or PSD95,markers for pre-or post-synaptic components,we found synaptophysin⁺and PSD95⁺puncta were present in GFP⁺microglia,colocalizing with lysosome marker Lamp1.Next,we systematically administered minocycline?Mino?to inhibit microglial engulfment of synapses,Mino-treated animals showed significantly higher freezing time,indicating blocking microglia phagocytosis inhibited memory forgetting.At the same time,by immunostaining,we found C1q was present within microglia,colocalizing with PSD95and CD68,a microglial lysosomal marker.Furthermore,we found the colocalization of C1q with the dendritic spines of engram cells.This suggests that microglia may eliminate synapses of engram cells in the DG.To test whether complement pathways are responsible for microglia-mediated engram dissociation and forgetting,we constructed a Cre-dependent AAV vector expressing CD55 to block complement pathways.We found that blocking complement pathways by expressing CD55specifically in engram cells prevented forgetting.This suggests that microglia mediate forgetting by complement-dependent synaptic elimination.We next examined whether microglia-mediated forgetting depends on the activity of engram neurons.We specifically expressed h M4Di in engram cells and inhibit their activity by treating the mice with CNO.35 days post training,the animals treated with CNO alone exhibited significantly decreased freezing.But PLX treatment prevented this facilitated forgetting,suggesting microglia eliminate synapses between engram cells,thus mediating engram dissociation and forgetting,in an activity-dependent manner.Finally,our study shows MEM-induced enhanced neurogenesis leads to increase synaptic engulfment by microglia,while depletion of microglia blocks memory forgetting induced by enhanced neurogenesis,suggesting that microglia contribute to neurogenesis-induced synaptic reorganization.Besides we found that depletion of microglia in the DG without neurogenesis or inhibition of complement pathways in CA1 engram cells prevents forgetting.This indicates that microglia-mediated synaptic reorganization is also happening in in non-neurogenic brain regions.In conclusion,our study suggests that complement-and activity-dependent elimination of synapses by microglia mediates dissociation of engram cells and forgetting of remote memory.