Fig. 8: BA fear neurons recruited during fear learning receive more monosynaptic vCA1 inputs conveying threat-predictive contextual information than other BA neurons do.

a Experimental setup for b–g. BA neurons active during fear conditioning (FC group) or those active in the home cages (HC group) were labeled with TVA-G-GFP. b Comparison of freezing behavior in Context A between groups 24 h after BA labeling. Mice in the FC group were fear conditioned in Context A and received saline (FC/SAL, 12 mice) or anisomycin injections (FC/ANI, 10 mice), whereas mice in the HC/ANI group remained in the home cages and received anisomycin injections (9 mice). ***p < 0.001, one-way ANOVA with post hoc comparisons. Mice in the FC/SAL group were not used for RV-mediated tracing experiments in c–g. c BA fear neurons in the FC group were labeled with TVA-G-GFP in a Cre-dependent manner under the control of the Arc promoter in the presence of 4-OHT in Arc-CreER^T2 mice. EnvA-ΔG-RV-mCherry infected TVA-G-expressing BA neurons, replicated, and propagated trans-synaptically to presynaptic neurons. d Neural circuit diagram showing trans-synaptic labeling of vCA1 neurons projecting to BA fear neurons labeled with TVA-G in the FC group (neuron D). EnvA-ΔG-RV-mCherry infected TVA-G-labeled BA neurons, resulting in mCherry expression in vCA1 neurons (neurons 1 and 4) that projected monosynaptically to BA fear neurons. Context A-encoding vCA1 neurons were immunostained for c-Fos (neuron 1). e Images showing TVA-G-GFP-labeled BA neurons (green) and RV-infected BA neurons expressing mCherry (red). f Images showing labeled vCA1 neurons in the FC (top) and HC groups (bottom). vCA1 neurons labeled with both mCherry and c-Fos are circled. g Comparison of the density of mCherry+ vCA1 neurons (left, p = 0.19; n.s., not significant), c-Fos+ cells (middle, p = 0.21), and the c-Fos+ proportion among all mCherry+ vCA1 neurons (right; **p = 0.001) between the FC (10 mice) and HC groups (9 mice). Two-sided unpaired t-tests were used. Dotted horizontal lines indicate the probability that a randomly selected vCA1 neurons is c-Fos+. h Experimental setup for i–l. i Mice were exposed to Context A to label vCA1 neurons active in Context A with ChR2. Mice were then fear conditioned in Context A to label BA fear neurons with tdTomato (tdT) and received anisomycin injections to prevent learning-induced synaptic strengthening in the vCA1–BA pathway. j Freezing behavior in Context A 24 h after fear conditioning (6 mice). k Representative traces of monosynaptic EPSCs induced by photostimulation of Context A vCA1 inputs and recorded in tdT− and tdT+ BA neurons in the presence of TTX and 4-AP as in Fig. 3m. l Plot of the average amplitude of EPSCs recorded in tdT− (16 neurons) and tdT+ neurons (16 neurons) as in k. ***p < 0.001, repeated measures two-way ANOVA. m BA fear neurons (F, red) receive more vCA1 inputs conveying threat-predictive contextual representations (blue) than other BA neurons (gray) do. Error bars represent the SEM. Source data are provided as a Source Data file. See also Supplementary Figs. 17–19.