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. 2022 Jun 1;42(22):4488–4504. doi: 10.1523/JNEUROSCI.1499-21.2022

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Figure 3. — BLA-BNST inactivation has opposing effects on freezing to a prolonged conditioned cue in male and female rats. A, Rats were conditioned as described previously (Fig. 1A), then injected with either saline (N = 21, black circles) or CNO (N = 22, gray circles) before testing. B, There was only a main effect of time (F_{(4.557,186.8)} = 81.06, p < 0.0001, two-way RM ANOVA) during prolonged cued fear training. There was no significant main effect of CNO treatment (F_(1,41) = 0.3874, p = 0.5371, two-way RM ANOVA), nor was there a main interaction between time and CNO (F_(10,410) = 0.4609, p = 0.9146, two-way RM ANOVA). C, During extinction, when all rats were grouped together, there was only a main effect of time (F_{(2.9868,121.7)} = 12.31, p < 0.0001, two-way RM ANOVA) on freezing behavior, but no main effect of drug alone (F_(1,41) = 0.1296, p = 0.7207, two-way RM ANOVA) nor interaction between drug and time (F_(10,410) = 0.3397, p = 0.9699, two-way RM ANOVA). D, For male rats that received either saline (N = 9, black circles) or CNO (N = 10, gray circles) injections before testing, there was only a main effect of time (F_(3.33,56.61) = 46.98, p < 0.0001, two-way RM ANOVA) on freezing during training, but no main drug effect (F_(1,17) = 0.06733, p = 0.7984, two-way RM ANOVA) nor significant interaction (F_(10,170) = 0.7074, p = 0.7167, two-way RM ANOVA). E, During testing, there was only a main effect of time (F_{(2.802,47.63)} = 3.528, p = 0.024, two-way RM ANOVA), but not of drug (F_(1,17) = 2.176, p = 0.1584, two-way RM ANOVA) nor an interaction (F_(10,170) = 1.143, p = 0.3331, two-way RM ANOVA). F, Female rats that were injected with either saline (N = 12, black circles) or CNO (N = 12, gray circles) had a main effect of time on freezing during training (F_{(4.794 105.5)} = 38.18, p < 0.0001, two-way RM ANOVA), but not for drug (F_(1,22) = 0.8739, p = 0.36, two-way RM ANOVA) nor an interaction between drug and time (F_(10,220) = 0.7041, p = 0.7201, two-way RM ANOVA). G, Similarly, during testing sessions, there was only a main effect of time (F_{(2.868,63.10)} = 10.75, p < 0.0001, two-way RM ANOVA), but not drug alone (F_(1,22) = 0.8374, p = 0.3701, two-way RM ANOVA) nor an interaction between time and drug (F_(10,220) = 1.239, p = 0.2673, two-way RM ANOVA). H, Total time spent freezing during 2 min baseline, the first 4 min of the prolonged tone (I), the second 4 min of the prolonged tone (J), and the postshock recovery period (K) of the testing session were separately analyzed. There was a significant interaction between drug and sex during the first half of the tone (H; F_(1,39) = 4.636, *p = 0.0375, two-way ANOVA) as CNO treatment reduced freezing in males, but increased freezing in females during the first half of the tone. There was no main effect of drug (F_(1,39) = 0.1044, p = 0.7483, two-way ANOVA) or sex (F_(1,39) = 0.6896, p = 0.4114, two-way ANOVA) alone.