The proteomic architecture of the synaptic engram supporting context memory

Sparsely distributed hippocampal CA1 engram cells, activated at the time of learning and receiving input from the CA3, are critical to contextual memory consolidation. This manifests as an augmentation of synaptic connectivity between these hippocampal subfields. Enhanced green fluorescent protein (GFP) reconstitution across synaptic partners (eGRASP) facilitates structural and electrophysiological analysis of input-specific synapses between engram cells. However, the protein landscape shaping and maintaining this enhanced engram synaptic connectivity remains unknown. Global proteomic analysis of the whole hippocampus results in population averaging, thereby diluting out effects specific to this sparse population of input-specific CA3-CA1 engram cell synapses. To address this challenge, we combined Fluorescence-activated synaptosome sorting (FASS) to isolate and harvest dorsal CA3 synapses onto CA1 engram cell synapses 72 hours after aversive contextual fear conditioning (CFC) or context exploration (CE). Utilizing ultrasensitive untargeted label-free quantitative mass spectrometry on these sorted synapses, we systematically identified 5500 unique proteins from sub-micron protein input and examined proteins enriched and depleted in CFC and CE engram cell synapses. Our findings unveil an enrichment of synaptic proteins associated with an upscaling of synaptic connectivity in CA1 engram cell synapses independent of context valence. In addition, specific postsynaptic protein signatures were enriched in engram cell synapses encoding context memory linked to negative valence, while neutral context representations exhibit greater presynaptic protein involvement. Validation through flow cytometry confirmed differential protein expression, with greater enrichment observed in an even smaller population of synapses between co-activated CA3 and CA1 engram cells during learning.