Figure 3. Behavioral responses indicate that recall is largest when using stimulating at the trough of theta.

(A) (Ai) Average freezing per epoch for FS+ (solid line) and FS- (dashed line) animals during habituation (gray) and during the four modes of stimulation (20 Hz: green, 6 Hz: yellow, peak: pink, trough: blue). ‘x’ indicates No Stim epochs. Shaded region represents 95% confidence interval. (Aii) Average increase in freezing using no stimulation (epochs 1 and 3) and stimulation (epochs 2 and 4). Only trough stimulation reliably caused increased freezing that resulted from activation of engram neurons (n=17 animals, paired t-test with Bonferroni correction). (B) (Bi) Average light-induced freezing was calculated for both experimental (FS+, shaded boxes) and control (FS-, open boxes) animals by subtracting epochs 2 and 4 from epochs 1 and 3, respectively, and averaging the value. Only 6 Hz stimulation and trough stimulation showed light-induced freezing that differed significantly from the non-foot shocked group. Light-induced freezing of using peak and 20 Hz stimulation was not significantly different than the control group (n=17 animals, independent t-test with Bonferroni correction; 6 Hz: *p=0.02 < 0.05; 20 Hz: p=0.6; peak: p=0.07; trough: ***p=0.0002 < 0.0001). (Bii) Light-induced freezing on the last day of habituation prior to the experiment only differed significantly for trough stimulation (n=17 animals, independent t-test with Bonferroni correction; 20 Hz: p=0.8, 6 Hz: p=0.4, peak: p=0.8, trough: *p=0.02 < 0.05). (C) Paired comparison between trough and peak stimulation for animals that exhibited light-induced freezing indicated significantly higher levels of freezing induced by trough stimulation (n=13 animals, **p=0.007 < 0.001). (D) Significantly higher freezing was observed upon exposure to the fearful context B 4 days after artificial reactivation of engram neurons in context A (n=17 animals, paired t-test, **p=0.01).