Fig. 10: Silencing context-specific vCA1 neurons during fear conditioning inhibited both contextual fear learning and synaptic potentiation of the vCA1–BA pathway.

a Experimental setup for b–g. AAV-DIO-ChR2-eYFP was injected into the vCA1, and AAV-TRE-mCherry was injected into the BA in Arc-CreER^T2 × Fos-tTA mice. b Mice were fed with doxycycline (Dox)-containing food (gray horizontal bar). Mice were exposed to Context A and received a 4-OHT injection for ChR2 expression in vCA1 neurons active in Context A. Mice were then taken off Dox for 48 h and fear conditioned in Context A for mCherry expression in BA fear neurons. Mice were put back on Dox immediately after fear conditioning and tested for freezing behavior in Context A on Day 2. Recording experiments (E-phys) were performed on Day 4. c Quantification of freezing responses in Context A on Day 2. n = 6 mice. d Left: vCA1 neurons active in Context A express CreER^T2 under the control of the Arc promoter, which then induced the recombination of the DIO in the presence of 4-OHT, resulting in permanent ChR2-eYFP expression. Right: images showing ChR2-eYFP-expressing vCA1 neurons (green). Red, Nissl stain. ACx, auditory cortex. e Left: BA neurons active during fear conditioning express tTA under the control of the c-Fos promoter, resulting in mCherry expression in the absence of Dox. Right: images showing mCherry+ BA neurons (red) and ChR2-eYFP-labeled vCA1 axons in the BA (green). f Traces of EPSCs recorded in mCherry (mCh)− and mCh+ BA neurons (inset; scale bar, 10 μm). EPSCs were induced by photostimulation of Context A vCA1 inputs and recorded in mCh− and mCh+ BA neurons. g Comparison of the AMPA/NMDA ratio between mCh− and mCh+ neurons (11 cells per group). ANOVA with post hoc comparisons was used to analyze combined data in g and n. h Experimental setup for i–n. Both AAV-DIO-hM₄D_i-mCherry and AAV-DIO-ChR2-eYFP were bilaterally injected into the vCA1, and AAV-TRE-mCherry was injected into the BA. i Mice were exposed to Context A and received a 4-OHT injection for the expression of both hM₄D_i and ChR2 in vCA1 neurons active in Context A. Mice were then taken off Dox and fear conditioned in Context A for mCherry expression in BA fear neurons. The mice received a CNO injection 30 min before fear conditioning to inhibit the activity of Context A vCA1 neurons. They were put back on Dox immediately after fear conditioning and tested for freezing behavior on Day 2. Recording experiments were performed on Day 4. j Quantification of freezing responses in Context A on Day 2. k Left: vCA1 neurons active in Context A expressed CreER^T2 under the control of the Arc promoter, which then induced the recombination of the DIO in the presence of 4-OHT, resulting in expression of both hM₄D_i-mCherry and ChR2-eYFP. Right: images showing vCA1 neurons (circles) expressing both hM4Di-mCherry (red) and ChR2-eYFP (green). l Left: BA neurons active during fear conditioning express tTA under the control of the c-Fos promoter, resulting in mCherry expression in the absence of Dox. Right: images showing mCherry+ BA neurons (red) and ChR2-eYFP-labeled vCA1 axons in the BA (green). m Traces of EPSCs induced by photostimulation of Context A vCA1 inputs and recorded in mCh− and mCh+ BA neurons (inset; scale bar, 10 μm) as in f. n Comparison of the AMPA/NMDA ratio between mCh− (10 cells) and mCh+ BA neurons (12 cells) (p = 1.00, two-way ANOVA with post hoc comparisons). Error bars represent the SEM. Source data are provided as a Source Data file. See also Supplementary Fig. 22.