Fig. 4: Stimulation frequency-dependent memory retrieval by activating engram cells.

a cFos⁺ neuron labeling using a cocktail of c-Fos-tTA and TRE-ChR2-eYFP. Wild-type mice raised on doxycycline (DOX) food were injected with the two viruses in the target regions. b Behavioral schedule. Beige shading signifies that mice were DOX, precluding ChR2-eYFP expression. Mice were taken off DOX 24 h before CFC. Natural recall test (Test). Optogenetic reactivation session in a neutral context B (Activation). c CA1 section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. d BLA section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. e LDTg section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. f AHN section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. g RE section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. h VLO section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. i AM section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. j PAG section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. k PVT section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. l V1 section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. m S1BF section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. n MD section (left). eYFP and ChR2-eYFP (n = 11 mice per group) groups. Statistical comparisons are performed using two-tailed unpaired t-tests; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM. P values: 0.0004 (4 Hz) (c), 0.0002 (4 Hz), 0.0011 (20 Hz) (d), 0.0105 (12 Hz) (e), 9.19E−05 (10 Hz), 0.0071 (20 Hz) (f), 0.0006 (4 Hz), 2.56E−06 (8 Hz) (g), 1.55E−05 (8 Hz), 0.0214 (20 Hz) (h), 0.0006 (4 Hz), 0.0004 (10 Hz) (i), 0.0085 (12 Hz), 0.0186 (20 Hz) (j), 0.0029 (4 Hz) (k). Source data are provided as a Source Data file.