A multiple Arc (mArc) tagging system to uncover the organizational principles of multiple memories

Engrams or memory traces are the neuronal ensembles that collectively store individual experiences. Genetic strategies based on immediate early genes (IEGs), such as Arc/Arg3.1 , allow us to tag the ensembles active during memory encoding and compare them to those active during retrieval. However, these strategies only allow for the tagging of one neural ensemble. Here, we developed a multiple Arc (mArc) system that allows for the tagging of two Arc + ensembles. We validated this system by investigating how context, time, and valence influence neuronal ensemble reactivation in the dentate gyrus (DG). We show that similar contextual and valenced experiences are encoded in overlapping DG ensembles. We also find that ensembles are modulated by time, where experiences closer in time are encoded in more similar ensembles. These results highlight the dynamic nature of DG ensembles and show that the mArc system provides a powerful approach for investigating multiple memories in the brain. HIGHLIGHTS ### Competing Interest Statement C.A.D. is named on provisional and non-provisional patent applications for the prophylactic use of (R,S) -ketamine and related compounds against stress-induced psychiatric disorders. M.S., T.D.Y., M.M., X.S., J.N., N.E.B, N.V., E.Z., and Y.L. declare no competing interests.