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. 2022 Apr 14;27(5):2580–2589. doi: 10.1038/s41380-022-01498-7

Fig. 4. Grin2b knockdown in GABAergic interneurons does not block the effect of NYX-783 on the inhibition of the spontaneous recovery of learned fear in male mice.

Fig. 4

a Representative images of Cre-dependent AAV2shGrin2b virus expression in IL mPFC in Gad1-Cre mice (Gad1-Cre_AAV2shGrin2b). Arrows indicate cells expressing both mCherry and EGFP. Arrowheads indicate Cre-recombined cells expressing only mCherry. b Representative traces of NMDA- and AMPA-induced inward currents before and after 0.1 µM NYX-783 in GABAergic interneurons. NMDA-induced inward current (n = 10 cells, 6 mice) and AMPA-induced inward current (n = 9 cells, 6 mice). Inward current (% of control) is shown as bars. Application of 0.1 µM NYX-783 significantly increased NMDA (10 µM)-induced inward currents but did not increase AMPA (5 µM)-induced inward currents in major GABAergic interneurons (Gad_tdTomato) in the IL mPFC. c Representative traces of NMDA- and AMPA-induced inward currents before and after 0.1 µM NYX-783 treatment. NMDA-induced inward current (n = 8 cells, 6 mice) and AMPA-induced inward current (n = 6 cells, 4 mice). Application of 0.1 µM NYX-783 did not increase the NMDA-induced inward current of GABAergic interneurons (Gad1-positive neurons) expressing Cre-dependent AAV2shGrin2b in the IL mPFC. AMPA-induced inward currents were not changed. b, c Two-tailed t test *p < 0.05. All the data are expressed as mean ± SEM. Bar = 100 µm. d Percentage of freezing in male mice after fear conditioning, extinction (day 1, day 2, day 3), and spontaneous recovery trials. e–h Within-group comparisons of male mice. e Black closed circles indicate saline injection with Cre-dependent AAV2shGrin2b virus expression in wild-type littermates (WT-shGrin2b_Saline). f Black open circles indicate NYX-783 injection with Cre-dependent AAV2shGrin2b virus expression in wild-type littermates (WT-shGrin2b_NYX-783). g Blue closed circles indicate saline injection with Cre-dependent AAV2shGrin2b virus expression in Gad1-Cre mice (Gad1-shGrin2b_Saline). h Blue open circles indicate NYX-783 injection with Cre-dependent AAV2shGrin2b virus expression in Gad1-Cre mice (Gad1-shGrin2b_NYX-783). n = 7–14 mice per group. d Conditioning and extinctions: three-way ANOVA. Spontaneous recovery: two-way ANOVA with Tukey’s multiple comparisons post hoc test. e–h Paired two-tailed t test *p < 0.05, ***p < 0.001. All the data are expressed as mean ± SEM. P preconditioned stimulus, CS conditioned stimulus, US unconditioned stimulus.