Fig. 3: Optogenetic manipulation of tone-responsive neurons in ACx modulates BLA engram cells to regulate DAFM.

A Schematic of virus injection. AAV9-c-fos-rtTA and AAV9-TRE-NpHR3.0-EGFP or AAV9-TRE-oChiEF-mcherry were infused into the ACx and representative confocal images of NpHR3.0 expression in the ACx. Scale bar: 200 μm. B Behavioral design. C, E, G, and I Schematic of optogenetic stimulation. D, F, H, J The percentage of the freezing time (1, 3) and the changing index (2, 4) in mice during 1 kHz retrieval stage (1, 2) or 10 kHz retrieval stage (3, 4) at day 3 when optogenetic inhibition (D, F) and activation (H, J) of the 10 kHz-tone responsive ACx neurons (D, H) and 1 kHz-tone responsive ACx neurons (F, J) (D NpHR3.0, n = 6, EGFP, n = 6; F NpHR3.0, n = 8, EGFP, n = 8; H oChiEF, n = 11, mCherry, n = 8; J oChiEF, n = 8, mCherry, n = 6). (K), Schematic of virus injection. AAV9-c-fos-tTA and AAV9-TRE-Gcamp6s were infused into the BLA, and AAV9-RAM-cre-ERT, AAV9-DIO-NpHR3.0-mcherry or AAV9- DIO-ChrimsonR-mCherry were infused into the ACx. Optical fiber was implanted in the ACx. Gcamp6s was injected to record neural activity of BLA engram cells by optical fiber. L Behavioral design. M–P The mean and SEM of the corresponding Ca²⁺ signal of engram cells in the BLA during 1 kHz retrieval stage (M–P, left) or 10 kHz retrieval stage (M–P right) at day 3 when inhibiting or activation of the 10 kHz-tone-responsive ACx neurons (M, O) or 1 kHz-tone-responsive ACx neurons (N, P). l n = 6; m, n = 7). The under red line indicated bins between the light-on and light-off trials, with two-tailed unpaired t-test significance (P < 0.05). Statistical tests: D, F, H, and J, freezing: two-way ANOVA with Bonferroni’s multiple comparisons tests; D, F, H, and J, changing index: unpaired two-tailed t-test. ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SEM.