Supplementary figure 1. The evoked network fEPSP was mediated by AMPA receptor
(a-b)Two mapped figuresshowing that the evoked baseline potentials from all active channels (a) were blocked by bath application of AMPA receptor antagonist GYKI53655 (GYKI, 100M)(b). The red S indicates the stimulation site. (c) The fEPSP slope (left) and the superimposed samples(right) from one channel showed that GYKI blocked the potential.(d) The summarized fEPSP slopes showed that GYKI blocked potentials from all 24 activated channels in one slice.(e) The summarized fEPSP slopes showed that GYKIblocked all activated potentials from 5 slices in 5 mice.
Supplementary figure 2. Inhibition of mGluR5or GSK3 in the ACC rescuedthe deficient acquisition and expression of trace fear memory in Fmr1 KO mice
(a) The schematic view of the microinjection and trace fear memory procedure. (b) Fmr1 KO mice with saline ACC injection exhibited a significantly lower freezing time during the training and testing session of fear memory test, compared with WT mice with saline ACC injection. However, MPEP (5 nM), SB 415286 (10, 50 nM, but not 100 nM) or CT99021 (10, but not 50 nM or 100 nM) microinjected into the ACC before conditioning significantly improved the freezing time both in the training and the testing sessions. #, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (c) Western blot samples (left panel) and summarized results (right panel) showing thatthe membrane GluA1 in the ACC of WT mice with saline injection was significantly upregulated when exposed to trace fear conditioning (p < 0.001, unpaired t-test, compared with WT mice with saline injection in control group). However, the membrane GluA1 of KO mice with saline injection exposed to trace fear conditioning was not upregulated. Meanwhile, MPEP (5 nM) or SB415286 (10 nM) injection into the ACC before trace fear conditioning rescued the upregulationof the membrane GluA1 in the ACC of KO mice. #, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (d) Western blot samples (left panel) and summarized results (right panel) showing that the expression of membrane GluA1 was significantly unregulated in KO mice with CT99021 (10 nM) injection, in comparison with KO mice with saline injection. Mice from all groups were exposed to trace fear memory training. ***, p < 0.001.
Supplementary figure 3. Time course of the freezing behavior in Fmr1 KO mice exposed to trace fear memory test
In saline treated groups (a), Fmr1 KO mice showedsignificantly reduced freezing performance from ITI-6 to ITI-10during tracefear training and from ITI-1 to ITI-10 during fear testing, compared with Fmr1WTmice(F(1, 270) = 80.24/140.90 in training/testing, p < 0.001; Two-way ANOVA followed by SNK’s post hoc test; *, p < 0.001). Microinjections of MPEP (5 nM) (b), SB 415286 (10 nM) (c) or CT99021 (10 nM) (d) into the ACC rescued the freezing responses of Fmr1 KO mice. During the training and testing period, there was no difference of the freezing responses between WT + MPEP and KO + MPEP groups (F(1, 130) = 1.97/3.80 in training/testing, p > 0.05), WT + SB415286 and KO + SB415286 groups (F(1, 130) = 3.80/2.77 in training/testing, p > 0.05) or WT + CT99021 and KO + CT99021 groups (F(1, 110) = 0.48/3.39 in training/testing, p > 0.05) (Two-way ANOVA).
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