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. 2020 Nov 20;9:e61882. doi: 10.7554/eLife.61882

Figure 5. Inhibiting CA1 neurons activated by the enriched environment experience prevents memory improvement by EE in Fmr1 KO mice.

(A) Experimental protocol for chemogenetic inhibition of EE-activated neurons before fear conditioning training. (B) Representative images showing EE-activated neural ensembles labeled with tdTomato (red) and fear conditioning learning-activated neurons labeled with FOS immunostaining (green) in control and inhibitory DREADD-expressing mice CA1. The circles highlight reactivated EE-engaged cells during fear conditioning learning (yellow). Scale bar: 100 μm. (C) Quantification of neuronal activation by EE (Kruskal-Wallis test: H (3)=0.1518, p=0.9850). (D) Quantification of neuronal activation by fear conditioning learning (Kruskal-Wallis test: H (3)=5.669, p=0.1289). (E) Quantification of neuronal activation during subsequent learning (FOS+) in EE-engaged (tdTomato+) and non-EE engaged (tdTomato-) populations [WT group: two-way ANOVA: group factor, F(1, 28)=9.608, **p<0.01; interaction, F(1, 28)=20.22, ***p<0.001; Tukey post hoc test: ***p<0.001; *p<0.05. KO group: two-way ANOVA: group factor, F(1, 28)=0.3268, p=0.5721; interaction, F(1, 28)=16.29, ***p<0.001; Tukey post hoc test: ***p<0.001; *p<0.05]. (F) Experimental protocol for EE-activated neural ensemble inhibition and contextual fear memory test. (G and H) Contextual fear conditioning test. Freezing levels were measured immediately after fear conditioning training (G) and again in training context 3 days after (H) (**p<0.01, two-tailed Mann-Whitney U test). n/N, number of mice/number of independent litters. All graphs represent mean ± SEM.

Figure 5.

Figure 5—figure supplement 1. Behavioral characterization of Fmr1 KO mice after inhibition of EE-activated neurons in CA1.

Figure 5—figure supplement 1.

(A and B) Conventional contextual fear conditioning test. Three pairs of tone (30 s, gray bar) and foot shock (2 s, red bar) were delivered with a 60 s interval. Learning curves (A) (two-way repeated measure ANOVA: group factor, F(3, 28)=0.5147, p=0.6755; interaction, F(33, 308)=1.176, p=0.2392) and freezing levels measured immediately after fear conditioning training (B) (p=0.1602, Kruskal-Wallis test). (C) Learning curve of contextual fear conditioning. The intensive fear conditioning training paradigm has three sessions, each consists of four pairs of co-terminating tone (20 s, gray bar) and foot shock (2 s, red bar). Freezing levels before, during, and after each tone-shock pair were quantified (Two-way repeated measure ANOVA: training 1: group factor, F(1, 19)=0.3120, p=0.5830; interaction, F(24, 456)=1.084, p=0.3575; training 2: group factor, F(1, 19)=0.7560, p=0.3954; interaction, F(24, 456)=1.304, p=0.1540; training 3: group factor, F(1, 19)=0.4480, p=0.5113; interaction, F(24, 456)=0.6499, p=0.8984). (D–G) Characterization of CNO’s effect on locomotion and freezing behavior. (D) Open field test. Left, quantification of track length in 30 min (p=0.5317, Mann-Whitney test); Right, quantification of percent track length in center (p=0.3217, Mann-Whitney test). (E–G) Conventional contextual fear conditioning learning. Three pairs of tone (30 s, gray bar) and foot shock (2 s, red bar) were delivered with a 60 s interval. Learning curves (E) (two-way repeated measure ANOVA: group factor, F(1, 8)=0.06812, p=0.8007; interaction, F(11, 88)=0.5860, p=0.8355) and freezing levels measured immediately after fear conditioning training (F) (p=0.4444, Mann-Whitney test). (G) Freezing level in training context measured 1 day after training (p=0.8016, Mann-Whitney test).