Extended Data Fig. 7: Artificial chemogenetic activation of LA CamK2α principal neurons.

a) All groups of mice - CamK2α. iPKR hM3Dq, CamK2α.hM3Dq and CamK2α WT, exhibited low freezing during ITI in LTM1. XY plots showing %freezing during individual ITIs and Post-CS (left; RM Two-way ANOVA) and bar graphs showing mean %freezing during ITI (right; One-way ANOVA). n = 6–9 per group. b) During LTM2, administration of DREADD agonist C21 caused an increase in freezing during ITI for both CamK2α.hM3Dq and CamK2α. iPKR hM3Dq groups compared to CamK2α WT mice. XY plots showing %freezing during individual ITIs and Post-CS (left). n = 5–7 per group. RM Two-way ANOVA genotype: F(2,15)=12.63, ***p=0.0006). Bar graphs showing mean %freezing during ITI (right). One-way ANOVA with Bonferroni’s post-hoc test. *p<0.05 and **p<0.01. c) CamK2α. eIF2α (A/A) mice displayed comparable learning in the diferential threat conditioning training for both CS+ (right) and CS- (left). d) However, in the LTM test, they displayed significant increase in CS- response compared to CamK2α WT mice (**p<0.01). Two-way ANOVA with Bonferroni’s post-hoc test. CS: F(1,28) = 49.18, ****p<0.0001; Genotype: F(1,28) = 15.26, ***p=0.0005. e) The cTD discrimination index was significantly lower for CamK2α. eIF2α (A/A) mice (**p<0.01) relative to controls. n=7–10 per group; Unpaired t-test. f) Besides stimulus generalization, CamK2α. eIF2α (A/A) mice also displayed cognitive inflexibility and could not stop freezing after the tone offset, and thus had significantly higher freezing rate during the ITIs. RM Two-way ANOVA with Bonferroni’s post-hoc test. Genotype: F(1,10) = 16.70, **p=0.0022. g) Mean freezing response during ITI is significantly increased in CamK2α. eIF2α (A/A) mice (**p=0.0097) . n=7-10 per group; Unpaired t-test. Data are presented as mean +/- SEM.