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

Fig. 3. Grin2b knockdown in glutamatergic neurons blocks the effect of NYX-783 on the inhibition of the spontaneous recovery of learned fear in male mice.

Fig. 3

a Top: schematic of the Cre-dependent Grin2b knockdown (AAV2shGrin2b) construct before and after Cre recombination to generate the active construct. Bottom: representative images of Cre-dependent AAV2shGrin2b virus expression in the IL mPFC in Camk2α-Cre transgenic mice (Camk2α-Cre_AAV2shGrin2b). Arrows indicate cells expressing both mCherry and EGFP. Arrowheads indicate Cre-recombined cells expressing only mCherry. b Camk2α_tdTomato (Camk2α-Cre::Ai14): representative traces of NMDA- and AMPA-induced inward currents before and after 0.1 µM NYX-783. NMDA-induced inward current (n = 16 cells, 8 mice) and AMPA-induced inward current (n = 8 cells, 7 mice). Bar graph (% of control): 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 of pyramidal neurons in the IL mPFC. c Camk2α_shGrin2b (Camk2α-Cre_ AAV2shGrin2b): representative traces of NMDA- and AMPA-induced inward currents before and after 0.1 µM NYX-783 treatment. NMDA-induced inward current (n = 6 cells, 4 mice) and AMPA-induced inward current (n = 7 cells, 4 mice). Application of 0.1 µM NYX-783 did not increase the NMDA-induced inward currents of Camk2α-positive neurons expressing Cre-dependent AAV2shGrin2b in the IL mPFC. AMPA-induced inward currents were not changed. Bar graph: NYX-783 (1 mg/kg; i.p.) treatment significantly inhibited spontaneous recovery only in WT mice infused with Cre-dependent AAV2shGrin2b virus, whereas this effect of NYX-783 was blocked in Camk2α-Cre mice after Grin2b knockdown. 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 Red closed circles indicate saline injection with Cre-dependent AAV2shGrin2b virus expression in Camk2α-Cre mice (Camk2α-shGrin2b_Saline). h Red open circles indicate NYX-783 injection with Cre-dependent AAV2shGrin2b virus expression in Camk2α-Cre mice (Camk2α-shGrin2b_NYX-783). d Conditioning and extinction: three-way ANOVA. Spontaneous recovery: two-way ANOVA with Tukey’s multiple comparisons post hoc test. e–h Paired two-tailed t test **p < 0.01. All the data are expressed as mean ± SEM. P preconditioned stimulus, CS conditioned stimulus, US unconditioned stimulus.