Fig. 2. Silencing LEC LRIPs in CA1 alters both context and object recognition memory.

A. Diagram of the experimental design. Gad2-Cre mice were injected with AAV^Cre to express GFP or PSAM in LEC. PSEM was delivered bilaterally to the CA1 region just prior to the training phase of memory tasks. B. Confocal image (5×) of coronal section from a Gad2-Cre mouse injected in LEC with an AAV^Cre expressing PSAM-2A-GFP, showing expression of GFP (green) in LEC (DAPI in blue). C. Scheme of contextual fear conditioning (see Methods). On day 1, mice were exposed to Context A, then given a tone followed by footshock. On day 2, mice were re-exposed to Context A. On day 3, mice were exposed to novel Context B, followed by a tone. PSEM was delivered just prior to training in mice expressing GFP (control) or PSAM in LRIPs. C. Bar plot (mean ± SEM) of time spent freezing (GFP, green; PSAM, purple): Day 1, in Context A before (Ctx A) and after (CS+US) footshock; Day 2, during recall testing in Context A; Day 3, in novel Context B before (Ctx B) and after cued tone (Day 3 CS.). Two-way repeated-measures ANOVA revealed no significant difference between groups in freezing on day 1 in Context A (treatment × time F (6, 105) = 0.8055, P = 0.5679; treatment F (1, 105) = 3.655, P = 0.0586; time F (6, 105) = 8.583, P < 0.0001). There was a significant difference in freezing between groups in Context A on day 2 (treatment × time F (4, 48) = 0.8918, P = 0.4761; treatment F (12, 48) = 5.069, P < 0.0001; time F (4, 48) = 11.75, P < 0.0001) and in Context B (no tone) on day 3 (treatment × time F (3, 45) = 1.230, P = 0.3069; treatment F (15, 45) = 2.246, P < 0.0186; time F (3, 45) = 53.01, P < 0.0001). The PSAM group showed significantly greater freezing on Day 3 in context B versus context A on Day 1 prior to footshock (treatment F (12, 24) = 5.332; time F (2, 24)=19.76; P < 0.0002). The GFP control group showed no significant difference in freezing in context A on Day 1 versus context B on Day 3 (treatment F (18, 18) = 0.4932; time F(2, 18) = 12.84; P = 0.928 n.s.). E. Schematic of experiment to test effect of silencing LEC LRIPs on novel object recognition (NOR). Mice were exposed to two objects in training trials 1 and 2, followed by a test trial in which one (now familiar or “old”) object was replaced by a novel (“new”) object. Prior to training, mice were infused with 0.5 µl of either 15 µM PSEM³⁰⁸ plus the dye miniRuby (silenced group, + PSEM) or miniRuby alone (control). Both groups expressed PSAM in LEC. F. Bar plots of time spent with familiar (old) versus novel (new) object in test trial. The PSEM treated group explored the old object for 49.38 ± 3.55 s (P < 0.005 versus control) and the new object for 86.38 ± 7.49 s (n = 6; P < 0.05, new versus old object, paired t-test). G. The discrimination index, calculated as (time spent exploring the new object – time spent exploring old object)/(total exploration time), was significantly greater in control versus PSEM-treated mice (P < 0.05, paired t-test).