Recently acquired memories are reactivated in the hippocampus during sleep, an initial step for their consolidation1–3. This process is concomitant with the hippocampal reactivation of previous memories4–6, posing the problem of how to prevent interference between older and recent, initially labile, memory traces. Theoretical work has suggested that consolidating multiple memories while minimizing interference can be achieved by randomly interleaving their reactivation7–10. An alternative is that a temporal microstructure of sleep can promote the reactivation of different types of memories during specific substates. Here, to test these two hypotheses, we developed a method to simultaneously record large hippocampal ensembles and monitor sleep dynamics through pupillometry in naturally sleeping mice. Oscillatory pupil fluctuations revealed a previously unknown microstructure of non-REM sleep-associated memory processes. We found that memory replay of recent experiences dominated in sharp-wave ripples during contracted pupil substates of non-REM sleep, whereas replay of previous memories preferentially occurred during dilated pupil substates. Selective closed-loop disruption of sharp-wave ripples during contracted pupil non-REM sleep impaired the recall of recent memories, whereas the same manipulation during dilated pupil substates had no behavioural effect. Stronger extrinsic excitatory inputs characterized the contracted pupil substate, whereas higher recruitment of local inhibition was prominent during dilated pupil substates. Thus, the microstructure of non-REM sleep organizes memory replay, with previous versus new memories being temporally segregated in different substates and supported by local and input-driven mechanisms, respectively. Our results suggest that the brain can multiplex distinct cognitive processes during sleep to facilitate continuous learning without interference.
