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. Author manuscript; available in PMC: 2021 Nov 10.
Published in final edited form as: Nat Neurosci. 2020 Aug 3;23(10):1229–1239. doi: 10.1038/s41593-020-0679-6

Extended Data Fig. 4. CNO administration during acquisition reduces CA1 and ACC activity at the time of remote recall only in GFAP∷hM4Di mice, and does not affect neuronal proliferation, differentiation, or survival (Related to Figure 2).

Extended Data Fig. 4

(A) Active neurons expressing cFos were quantified in the CA1, ACC, dentate gyrus (DG), retrosplenial cortex (RSC), and basolateral amygdala (BLA). GFAP∷hM4Di mice from figure 2A,B that were injected with CNO (n=6) before fear conditioning and showed impaired remote recall compared to Saline controls (n=6), also demonstrated reduced number of cFos expressing neurons in CA1 and ACC (p<0.05 for both)(B). No changes in cFos expression in the DG or RSC were observed in these mice, but the reduced fear was accompanied by a significant reduction in cFos expression in the BLA (p<0.05)(C). GFAP∷eGFP control mice were injected with CNO (n=5) or Saline (n=5) before fear conditioning, and then tested on the next day. No changes were observed in recent memory (D) or in the number of neurons active during recent recall in the CA1 or ACC (E). Other GFAP∷eGFP mice were injected with CNO (n=5) or Saline (n=6) before fear conditioning, and then tested on the next day and again 21 days later. No changes were observed in recent or remote memory (F), or in the number of neurons active during remote recall in the CA1 or ACC (G). Representative images of GFAP∷eGFP (green) and cFos (red in H,J green in I,K) following recent (H-I) or remote (J-K) recall in the CA1 (H,J) and ACC (I,K) are presented. (L) GFAP∷eGFP mice were injected with CNO or Saline together with BrdU before fear conditioning, and then tested on the next day. No changes were observed in stem cell proliferation (Brdu in white)(M) or in the number of young, Doublecortine (DCx)-positive neurons (white)(N). (O) GFAP∷eGFP mice were injected with CNO or Saline and BrdU before fear conditioning, and then tested 21 days later. No changes were observed in stem cell proliferation and differentiation (P) or in the number of young, DCx-positive neurons (Q). Scale bars = 100μm for CA1, ACC and whole DG, 10 μm for zoomed-in cells. Data presented as mean ± SEM.