Fig. 7

Optogenetic manipulation of the ACC-BLA projection changes freezing response to an innate threat. a Illustration of eNpHR3.0-based inhibition strategy of the ACC terminals in the amygdala. b Representative confocal images showing eNpHR3.0-eYFP terminals in the amygdala. Scale bar, 500 µm. c Experimental procedure for fox urine test with photoinhibition of ACC-amygdala projection. d–f Percentage of time spent freezing during the fox urine test was selectively increased by photoinhibition in the eNpHR3.0 (ACC projection) group (n = 10) (d), but not in the eGFP (ACC projection) group (n = 10) (e) or eNpHR3.0 (PL projection) group (n = 7) (f). Wilcoxon matched-pair rank tests for eNpHR3.0 ACC projection (P = 0.0020, = 0.0039, = 0.019). Paired t-tests for eGFP ACC projection (P = 0.1736, = 0.4154, = 0.1171) and eNpHR3.0 PL projection (P = 0.0024, = 0.0181, = 0.0016). g Light-induced change in percentage of freezing behavior for d–f, calculated by subtracting freezing values within the first OFF–ON cycle. One-way ANOVA (F_{(2, 24)} = 19.65, P < 0.0001) and Tukey’s post hoc test (eNpHR3.0 ACC projection vs. eGFP ACC projection, P < 0.0001; eNpHR3.0 ACC projection vs. eNpHR3.0 PL projection, P < 0.0001; eGFP ACC-projection vs. eNpHR3.0 PL-projection, P = 0.7925). h Experimental procedure for fox urine test with ChR2-based photoactivation of ACC terminals in the amygdala. i, j Percentage of time spent freezing during the fox urine test was decreased in the ChR2 group (n = 8) (i) but not in the eGFP group (n = 7) (j). Paired t-tests for ChR2 group (P = 0.0007, = 0.0815, = 0.0488) and eGFP group (P = 0.5612, = 0.6859, = 0.5114). k Light-induced change in percentage of freezing behavior for i and j, calculated by subtracting freezing values within the first OFF–ON cycle. Unpaired t-test (P = 0.0008, t = 4.370, df = 13). Line graphs are expressed as mean ± s.e.m. Box-whisker plot is expressed as median, interquartile range with 5-95 percentile distribution. *P < 0.05, **P < 0.01, ***P < 0.001