Figure 2. LB affects the short-term expression of fear during early development.
(A) Table of the postnatal day at which each cohort underwent different phases of auditory cue conditioning. Different cohorts were used for each age group. (B) Schematic of auditory fear conditioning protocol. (C) Graphs of the percent time female (top) and male (bottom) mice spent freezing (immobile) during the conditioning and recall test. LB female (t21 = 2.79, p=0.010) and male (t20 = 3.39, p=0.0028) mice exhibited decreased fear expression at PND 22 when compared to age and sex matched controls, an effect not observed at other ages (Females: PND 17 (t20 = 1.43, p=0.16), 29 (t19 = 0.032, p=0.97), 51 (t21 = 0.73, p=0.47)) (Males: PND 17 (t24 = 0.68, p=0.50), 29 (t21 = 0.014, p=0.98), 51 (t19 = 1.42, p=0.16)). During auditory cue conditioning in females, a main effect of rearing condition was observed at PND 28 (F(1,19) = 7.70, p=0.012), but not at 16 (F(1,20) = 0.52, p=0.47), 21 (F(1,21) = 3.32, p=0.082), or 50 (F(1,21) = 1.33, p=0.26). Post-hoc analysis revealed that PND 28 LB females had significantly lower levels of freezing on the 5th (t114 = 3.50, p=0.0039) and 6th (t114 = 3.80, p=0.0014) tones. A main effect of tone during conditioning was observed in females at all ages (PND 16: F(5,100) = 2.70, p=0.024; PND 21: F(5,105) = 4.11, p=0.0019; PND 28: F(5,95) = 39.3, p<0.0001; PND 50: F(5,105) = 104.1, p<0.0001), indicating that female mice learned the tone/foot shock association at all ages tested. No interaction between tone trial and rearing condition where observed at PND 16 (F(5,100) = 1.51, p=0.19) or 21 (F(5,105) = 0.83, p=0.52); however, significant interactions where observed at PND 28 (F(5,95) = 3.79, p=0.0035) and 50 (F(5,105) = 2.45, p=0.037). During auditory cue conditioning in males, no main effect of rearing condition was observed at any age tested (PND 16: F(1,24) = 0.15, p=0.69; PND 21: F(1,20) = 3.32, p=0.083; PND 28: F(1,21) = 0.78, p=0.38; PND 50: F(1,19) = 0.0017, p=0.96). However, a post-hoc analysis revealed that PND 21 LB males had significantly lower levels of freezing on the 5th (t120 = 3.25, p=0.0088) and 6th (t120 = 2.78, p=0.036) tones when compared to ctrl males. During conditioning in males a main effect of tone trial was observed at all ages (PND 16: F(5,120) = 3.33, p=0.0074; PND 21: F(5,100) = 12.14, p<0.0001; PND 28: F(5,105) = 54.63, p<0.0001; PND 50: F(5,95) = 62.84, p<0.0001), indicating that male mice learned the tone/foot shock association at all ages tested. No interaction between tone trial and rearing condition where observed in male conditioning at PND 16 (F(5,120) = 0.42, p=0.83), 28 (F(5,105) = 0.59, p=0.70), or 50 (F(5,95) = 0.49, p=0.77); however, a significant interaction was observed at PND 21 (F(5,100) = 4.05, p=0.0022). (Females: Ctrl n = 11, 8, 12, 11; LB n = 11, 15, 9, 12) (Males: Ctrl n = 16, 9, 12, 12; LB n = 10, 13, 11, 12). For effects on memory recall, following matching for conditioning curves see Figure 3—figure supplement 4. Additional tests for somatosensation were conducted to ensure that differences in freezing were not due to differences in foot-shock sensitivity, see Figure 2—figure supplement 2. Bars represent group means + / - SEM. Two-way repeated measure ANOVA followed by a Sidak’s multiple comparison analysis was used to analyze the conditioning curves. Unpaired two-tailed student t-tests were used to analyze differences in recall tests. *=p < 0.05, **=p < 0.01, ***=p < 0.001.
Figure 2—figure supplement 1. Effects on recall at PND 21 are not a consequence of conditioning deficits.
