Fig. 7: Dual independent labeling revealed selective strengthening of synapses that connect context-specific vCA1 neurons to BA fear neurons in contextual fear conditioning.

a Experimental setup. b Left: mice were taken off Dox and exposed to Context A to induce ChR2 expression in Context A vCA1 neurons. Mice received two additional vCA1 labeling sessions with a 1-week interval (vCA1 label 3×). Mice were put back on Dox immediately after each context labeling session. After tamoxifen injection, the mice were fear-conditioned in Context A on Day 1 for mCherry expression in BA fear neurons (BA label). The mice were tested for freezing behavior on Day 2 (Recall). Recording experiments (E-phys) were performed on Day 4. Right: quantification of freezing responses in Context (Ctx) A on Day 2 (n = 5 mice). c Left: vCA1 neurons active in Context A express tTA under the control of c-Fos promoter, resulting in ChR2-eYFP expression in the absence of Dox. Right: images showing ChR2-eYFP-expressing vCA1 neurons (green, circles). Red, Nissl stain. d Left: BA neurons active during fear conditioning express CreER^T2 under the control of c-Fos promoter, resulting in tamoxifen-dependent recombination of the DIO and subsequent mCherry expression. Right: images showing mCherry-expressing BA neurons (red, left) and eYFP-labeled vCA1 axons (green, right) in the BA. e Traces of EPSCs recorded in mCherry (mCh)− and mCh+ BA neurons. mCh+ neurons were identified with red fluorescence within the BA (inset; scale bar, 10 μm). EPSCs were induced with photostimulation of Context A vCA1 inputs and recorded as in Fig. 4d. f Left: comparison of the AMPA/NMDA (A/N) ratios between mCh− and mCh+ BA neurons (two-sided paired t-test). Right: scatter plot of the A/N ratios in 12 pairs of mCh− (x-axis) and mCh+ BA neurons (y-axis) that were adjacent to each other. Error bars represent the SEM. Source data are provided as a Source Data file. See also Supplementary Fig. 16.