Fig. 1: Ensembles activated during prelearning sleep are recruited into the upcoming engram.

A Schematic diagram showing the miniature microscope placed at the hippocampal CA1 in c-fos-tTA x G-CaMP-7 double transgenic mice injected with TRE-KikGR lentivirus for labeling and visualizing both engram and nonengram cells. B Experimental design for the calcium imaging and behavior paradigm. NREM (N1) and REM (R1) in prelearning sleep sessions, and then learning (A), are followed by postlearning sleep sessions NREM (N2) and REM (R2) on day 1. On day 2, a snapshot for KikGR is taken, and then after ~2 h, a retrieval session (a’) was followed by a different context (B) (top), Representative snapshots of KikGR (scale bar, 100 µm) taken before learning (1), 24 h after learning (2), and after photoconversion to render KikGR invisible to the microscope (3) (bottom). This was done for all mice that performed the imaging paradigm (n = 6 mice). C Representative matching score (MS) matrixes for engram (top) and nonengram (bottom) cells across sessions to show similarity between ensembles. D Normalized MS analysis in reference to session A for both engram (green) and nonengram (blue) cells across sessions. n = 6 mice for engram and nonengram. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical comparisons were made using Repeated-measures two-way ANOVA with Bonferroni’s multiple comparisons test. Data represent the mean ± s.e.m. The Experiment was independently repeated six times. Source data are provided as a Source Data file. Detailed statistics are shown in Supplementary Data 1.