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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

Figure 4. Gi pathway activation in CA1 astrocytes during memory acquisition specifically prevents the recruitment of CA1 neurons projecting to ACC.

Figure 4

(A) AAV-retro-CaMKII∷Cre was injected into the ACC, and AAV5-ef1α∷DIO-GFP together with AAV8-GFAP∷hM4Di-mCherry were injected into CA1. (B) Together, these three vectors induced the expression of GFP (green) in CA1 neurons projecting to the ACC, and hM4Di (red) in CA1 astrocytes. (C) GFP-positive axons of CA1 projection neurons are clearly visible in the ACC. (D) Mice expressing GFP in ACC-projecting CA1 neurons and hM4Di in their CA1 astrocytes that were injected with CNO (n=8) or Saline (n=7) 30 minutes before FC showed similar immediate freezing following shock administration. (E) Fear-conditioned mice showed increased cFos levels in the CA1 compared to home-caged mice (p<0.05), with no effect for CNO administration. cFos levels in the ACC were increased in mice that underwent conditioning after being injected with Saline (p<0.05), but not in CNO-injected mice. (F) Fear-conditioned mice injected with Saline showed an >130% increase in the percent of CA1 cells projecting into the ACC that express cFos, compared to home-caged mice (p<0.05). CNO administration completely abolished the recruitment of these cells during learning. Representative images of hM4Di in astrocytes (red), GFP in ACC-projecting CA1 neurons (green) and cFos (pink) in the CA1 of Saline- (G) or CNO- (H) injected mice are presented. (I) AAV-retro-CaMKII∷Cre was injected into the NAc, and AAV5-ef1α∷DIO-GFP together with AAV8-GFAP∷hM4Di-mCherry were injected into CA1. (J) Together, these three vectors induced the expression of GFP (green) in CA1 neurons projecting to the NAc, and hM4Di (red) in CA1 astrocytes. (K) GFP-positive axons of CA1 projection neurons are clearly visible in the NAc. (L) Mice expressing GFP in NAc-projecting CA1 neurons and hM4Di in their CA1 astrocytes that were injected with CNO (n=10) or Saline (n=8) 30 minutes before FC showed similar immediate freezing following shock administration. (M) Fear-conditioned mice showed increased cFos levels in the NAc compared to home-caged mice (p<0.05), with no effect for CNO administration. (N) Fear-conditioned mice injected with either Saline or CNO showed an >60% increase in the percent of CA1 cells projecting into the NAc that express cFos, compared to home-caged mice (p<0.05). CNO administration had no effect on the recruitment of these cells during learning. All scale bars=50μm. Data presented as mean ± SEM.