Memory engram synapse 3D macromolecular architecture visualized by cryoCLEM-guided cryoET.

Memory is incorporated into the brain as physicochemical changes to engram cells. These neuronal populations form complex neuroanatomical circuits, are modified by experiences to store information, and allow memory recall. At the molecular level, learning modifies synaptic communication to rewire engram circuits. How macromolecules are organized within engram synapses is unknown. Here, we establish engram labeling technology combined with cryogenic correlated light and electron microscopy (cryoCLEM)-guided cryogenic electron tomography (cryoET) to visualize the in-tissue 3D macromolecular architecture of engram synapses of a contextual fear memory within the mouse hippocampus. Engram synapses exhibited structural diversity of macromolecular constituents and organelles in both pre- and postsynaptic compartments and within the synaptic cleft, including in membrane proteins, synaptic vesicle occupancy, and F-actin copy number. This "engram to tomogram" approach, harnessing in vivo functional neuroscience and structural biology, provides a methodological framework for testing fundamental molecular plasticity mechanisms within engram circuits.