Investigation of synaptic connectivity in functional in vitro neuronal assemblies.

Studies using genetic tagging and optogenetics demonstrated that reactivation of memory engrams, neuronal ensembles encoding specific learned information, can trigger memory recall and that synaptic potentiation among engram neurons is critical for memory persistence. However, the complexity of intact brain networks has limited mechanistic access to the processes underlying engram formation. Here, we introduce a hybrid in vitro platform that recapitulates, in a simplified and controllable setting, the core principles used in vivo to activate engrams. By combining digital light processing (DLP) with optogenetics, we imposed Hebbian co-activation of two targeted neurons, inducing the emergence of a functional cell assembly module. This artificial co-firing produced synaptic strengthening and spatial clustering of potentiated spines along the dendrites connecting the co-activated neurons, hallmarks of engram connectivity. Our system provides a reductionist yet biologically relevant framework to dissect, with high spatial and temporal resolution, the cellular and molecular determinants of cell assembly formation.