Fig. 1: Identification of ACC pyramidal neurons encoding observational fear.

a Schematic of observational fear conditioning (OFC). During habituation, the observer (OB) and demonstrator (DM) mice were individually placed in separate chambers and allowed to explore for 5 min. During conditioning, the OB mouse observed the DM mouse receiving foot shocks through a transparent partition for 4 min. b Illustration of microendoscopic Ca²⁺ imaging in the right hemisphere of ACC. c Representative image showing GCaMP6f expression in individual cell bodies and the GRIN lens tract above the ACC. Green, GCaMP6f; blue, DAPI. Scale bar, 50 μm. d Example field of view image showing raw Ca²⁺ fluorescence signals. e Schematic of three behaviors – OB-freezing, DM-reaction, and DM-freezing – during OFC. f Example denoised Ca²⁺ traces from cells responsive to OB-freezing (pink), DM-reaction (blue), and DM-freezing (yellow) in d. g Receiver operating characteristic (ROC) curves from example neurons for OB-freezing that are excited (auROC = 0.79), inhibited (auROC = 0.28), and non-responsive (auROC = 0.50). h Venn diagrams illustrating neurons responding to OB-freezing, DM-reaction, and DM-freezing. i Example field of view from d depicting the spatial distribution of cells encoding the three behaviors during OFC. j Pairwise distances among different subsets of cells (Data) were compared to those from randomly shuffled behavior labels (Shuffled). Sample size of OB-freezing = 2,933; DM-reaction = 955; DM-freezing = 1,715. The pairwise distance among cells encoding DM-reaction was significantly reduced compared to the shuffled data (Two-sided unpaired t-test with Welch’s correction, t = 3.437, df = 1008, p = 0.0006). k Heatmap of example neurons and trial-averaged responses from OB-freezing, DM-reaction, and DM-freezing cells around behavioral onsets. l Proportions of excited and inhibited cells corresponding to each of the three behaviors. n = 12 mice. m Example Ca²⁺ traces of excited and inhibited individual neurons selectively responding to OB-freezing (pink), DM-reaction (blue), or DM-freezing (yellow) behaviors. Colored blocks indicate each behavior bout. Source data are provided as a Source Data file. P-values in the graphs are indicated as follows, *** p < 0.0001. All data are presented as means ± S.E.M.