Fig. 4: Stress-activated neurons in the PVT modulate safety learning.

A Mice injected with c-Fos-tTA and TRE-GCaMP6 virus, with an optical fiber implemented into the PVT. B Experimental procedure for labeling stress-response neurons in the PVT. C Representative image showing the expression of GCaMP6 in stress-activated neurons in PVT. Scale bar: 100 μm. D Averaged Ca²⁺ responses in the PVT stress-activated neurons during CS− and CS+ after fear conditioning (two-tailed paired t-test, t = 0.3114, df = 5; N = 6 mice, CS- vs. CS+, P = 0.7681). E Averaged Ca²⁺ responses in the PVT stress neurons during CS− and CS+ after safety learning (two-tailed paired t-test, t = 6.542, df = 5; N = 6 mice, CS- vs. CS+, P < 0.01). F Schematics for injections of c-Fos-tTA and TRE-ChR2 virus in the PVT (left) and experimental procedure (right). G Freezing levels (left) and discrimination index (right) during CS- retrieval before, during, and after opto-stimulation of PVT stress-activated neurons (one-way ANOVA, F(2,10) = 37.32, P < 0.0001; N = 6 mice, test 1(laser off)vs. test 2 (laser on), P < 0.001, test 2 (laser on)vs. test 3 (laser off), P < 0.0001, Bonferroni’s post-test; one-way ANOVA, F(2,10) = 21.13, P < 0.001; N = 6 mice, test 1(laser off)vs. test 2(laser on), P < 0.01, test 2 (laser on)vs. test 3 (laser off), P < 0.001, Bonferroni’s post-test). H Schematics for injections of c-Fos-tTA and TRE-NpHR virus in the PVT (left) and experimental procedure (right). I Freezing levels (left) and discrimination index (right) during CS- retrieval before, during and after opto-stimulation of PVT stress-activated neurons (one-way ANOVA, F(2,12) = 0.1201, P = 0.8879; N = 7 mice, test 1 (laser off) vs. test 2 (laser on), P > 0.99, test 2 (laser on) vs. test 3 (laser off), P > 0.99, Bonferroni’s post-test; one-way ANOVA, F(2,12) = 0.7088, P = 0.5117; N = 7 mice, test 1 (laser off) vs. test 2 (laser on), P > 0.99, test 2 (laser on) vs. test 3 (laser off), P > 0.99, Bonferroni’s post-test).