Hippocampal engram networks for fear memory recruit new synapses and modify pre-existing synapses in vivo

As basic units of neural networks, ensembles of synapses underlie cognitive functions such as learning and memory. These synaptic engrams show elevated synaptic density among engram cells following contextual fear memory formation. Subsequent analysis of the CA3-CA1 engram synapse revealed larger spine sizes, as the synaptic connectivity correlated with the memory strength. Here, we elucidate the synapse dynamics between CA3 and CA1 by tracking identical synapses at multiple time points by adapting two-photon microscopy and dual-eGRASP technique in vivo. After memory formation, synaptic connections between engram populations are enhanced in conjunction with synaptogenesis within the hippocampal network. However, extinction learning specifically correlated with the disappearance of CA3 engram to CA1 engram (E-E) synapses. We observed “newly formed” synapses near pre-existing synapses, which clustered CA3-CA1 engram synapses after fear memory formation. Overall, we conclude that dynamics at CA3 to CA1 E-E synapses are key sites for modification during fear memory states. •Hippocampal synaptic connections are tracked by two-photon imaging of dual-eGRASP•E-E synapses undergo synaptogenesis according to fear conditioning•Extinction learning significantly correlated with the disappearance of E-E synapses•Spatial distribution of new E-E synapses induces clustering of engram synapses Lee et al. use four-color two-photon imaging and dual-eGRASP system to enable longitudinal tracking of the synaptic connections between CA3 and CA1. From these data, they demonstrate a correlation between the dynamics of engram synapses and fear memory states. A particular distribution of newly formed synapses leads to clustering of E-E synapses.