Excitability mediates allocation of pre-configured ensembles to a hippocampal engram supporting contextual conditioned threat in mice

Little is understood about how engrams, sparse groups of neurons that store memories, are formed endogenously. Here, we combined calcium imaging, activity tagging, and optogenetics to examine the role of neuronal excitability and pre-existing functional connectivity on the allocation of mouse cornu ammonis area 1 (CA1) hippocampal neurons to an engram ensemble supporting a contextual threat memory. Engram neurons (high activity during recall or TRAP2-tagged during training) were more active than non-engram neurons 3 h (but not 24 h to 5 days) before training. Consistent with this, optogenetically inhibiting scFLARE2-tagged neurons active in homecage 3 h, but not 24 h, before conditioning disrupted memory retrieval, indicating that neurons with higher pre-training excitability were allocated to the engram. We also observed stable pre-configured functionally connected sub-ensembles of neurons whose activity cycled over days. Sub-ensembles that were more active before training were allocated to the engram, and their functional connectivity increased at training. Therefore, both neuronal excitability and pre-configured functional connectivity mediate allocation to an engram ensemble. [Display omitted] •Active neurons in hours before experience are preferentially allocated to engram ensemble•Active neurons in hours before experience are necessary for subsequent memory retrieval•Functionally connected neuronal ensembles detected days before conditioning event•Learning modifies functional connectivity between neurons in engram ensembles Neurons are allocated to an engram ensemble during an event. Mocle et al. found that engram neurons showed endogenously elevated activity hours before an event, but pre-existing functional connectivity days before an event, suggesting that small sub-ensembles of active neurons are allocated as groups and that their functional connectivity is modified by learning.