Fig. 3: Activity-dependent functional labeling identified vCA1 neurons that are active in a new context and project to a subset of BA neurons.

a Experimental setup for b and c. vCA1 neurons projecting to the BA were labeled with mCherry, whereas vCA1 neurons active in a novel context were labeled with eYFP in Fos-CreER^T2 mice. b Top: mice were exposed to a novel context (red vertical bars) after tamoxifen injection (Tam). Bottom: magnified images of the vCA1. A vCA1 neuron labeled with both eYFP and mCherry is circled. c The proportion of eYFP-labeled neurons among all mCherry+ vCA1 neurons. n = 5 mice. d Experimental setup for e and f. e Top: after tamoxifen injection, mice were exposed to Context A to label with tdTomato vCA1 neurons active in Context A. Mice were then exposed to Context A (A–A group) or Context B (A–B group) before brain fixation for c-Fos immunostaining (IHC). Middle and bottom: images showing vCA1 neurons labeled with tdTomato (red) or c-Fos (green). vCA1 neurons labeled with both tdTomato and c-Fos are circled. f Comparisons of tdTomato+ cell density (p = 0.69), c-Fos+ cell density (p = 0.53), Fos+ and tdTomato+ cell density (*p = 0.015), and c-Fos+ proportion among all tdTomato+ vCA1 neurons (**p = 0.007). n = 6 mice per group. Two-sided paired t-tests were used. g Experimental setup for h–n. Horizontal lines indicate the axons of vCA1 neurons, and vertical lines indicate the dendrites of BA neurons. vCA1 neurons active in Context A expressed ChR2-eYFP (blue). Photostimulation activated ChR2-expressing axons and induced postsynaptic responses recorded in BA neurons (Rec). h Top: after surgery, mice received a tamoxifen injection and were exposed to Context A as in b to induce ChR2-eYFP expression in vCA1 neurons active in Context A. They received three context labeling sessions with a 1-week interval. Bottom: images showing ChR2-eYFP+ vCA1 neurons (green) and their axons in the amygdala. Red, Nissl stain. i The proportion of ChR2-eYFP+ cells among all DAPI+ vCA1 neurons. n = 8 mice. j Representative traces of EPSCs induced by photostimulation of ChR2+ vCA1 axons and recorded at –80 mV in voltage-clamp mode in a BA principal neuron (red). EPSCs were inhibited by NBQX and MK-801 (black). Inset: image of BA neurons loaded with biocytin during recording and labeled with streptavidin-Alexa Fluor 633. k Left: EPSCs recorded in four BA neurons in a brain slice and induced by photostimulation of the same intensity. Right: scatter plot of the peak amplitudes of EPSCs recorded in BA neurons in each brain slice (n = 15 slices). Open circles indicate EPSC amplitudes in individual BA neurons. The average amplitude of EPSCs recorded in BA neurons in the same brain slice (black curve) was used to sort data along the x-axis in increasing order. l Histogram showing the distribution of the peak amplitudes of EPSCs induced by photostimulation of the same intensity (20.0 mW/mm²). n = 190 BA neurons. m Tetrodotoxin (TTX, 1 μM) completely blocked EPSCs in the Context A vCA1–BA pathway (left and middle). Subsequent application of 4-aminopyridine (4-AP, 1 mM) in the presence of TTX rescued EPSCs (right), indicating the monosynaptic nature of EPSCs. n Scatter plot of the peak amplitudes of monosynaptic EPSCs induced by photostimulation of the same intensity and recorded in BA neurons in each brain slice as in m. Open circles indicate EPSC amplitudes in each BA neurons. The average amplitude of EPSCs recorded in BA neurons in the same brain slice (black curve) was used to sort data along the x-axis in increasing order. Error bars represent the SEM. Source data are provided as a Source Data file. See also Supplementary Figs. 4–7.