Chemogenetic silencing of vGlut1+ DH→ RSC terminals impair encoding and retrieval of contextual fear conditioning. (a) Experimental design similar to Figure 1 except for infusion of a Cre-dependent AAV8-DIO-hM4D(Gi)-mCherry. Freezing during the context test was significantly reduced in vGlut1-Cre mice injected with CNO when compared to Veh (Veh: 61.5 ± 4.68; CNO: 37.13 ± 6.99; t = 2.87, P < 0.05 (n = 8/group)), but not in vGlut2-Cre mice (Veh: 49.48 ± 5.32; CNO: 45.66 ± 6.41; t = 0.45, P = 0.65; Veh n = 12, CNO n = 14). (b) Within-subject design was used to determine the effect of pretraining CNO on recent and remote memory. Significant treatment effects were found for each genotype (vGlut1-Cre: F1,16 = 43.78, P < 0.001; n = 9/group; vGlut2-Cre: F1,16 = 10.91, P < 0.01; Veh n = 12, CNO n = 14). However, vGlut1-Cre mice receiving CNO before training showed reduced freezing both at recent (Veh: 54.3 ± 6.36; CNO: 7.56 ± 2.29; P < 0.05) and remote memory tests (Veh: 55.8 ± 6.09; CNO: 22.7 ± 5.79; P < 0.01), whereas similarly treated vGlut2-Cre mice showed freezing deficits only at the remote (Veh: 61.3 ± 6.35; CNO: 30.9 ± 5.96; P < 0.01), but not recent test (Veh: 58.2 ± 3.86; CNO: 57.7 ± 5.06). (c) Infusion of CNO before the recent memory test impaired freezing to the conditioning context in vGlut1-Cre mice (Veh: 64.6 ± 3.08; CNO: 37.2 ± 6.98; t = 3.14, P < 0.01; n = 9/group) without affecting freezing in vGlut2-Cre mice (Veh: 47.5 ± 7.73; CNO: 53.9 ± 8.46; t = 0.56, P = 0.58; n = 7/group). Infusion of CNO before the remote memory test was ineffective in either mouse line (vGlut1-Cre, Veh: 36.2 ± 5.04; CNO: 38.2 ± 6.32; t = 0.61, P = 0.54; n = 15/group; vGlut2-Cre: Veh: 49.9 ± 8.62; CNO: 54.8 ± 8.78; t13 = 0.39, P = 0.7; n = 7/group).