Fig. 6

Inhibition of wFMNs-projecting vlPAG excitatory neurons improves the depressive-like behavior induced by CRS. a Schematic of experimental design. b Scheme for specific infection of wFMNs-projecting vlPAG excitatory neurons with hM4Di or mCherry by stereotaxic infusion of rAAV-retro-CaMKIIα-EGFP-Cre into the unilateral lFN at the site of wFMNs and AAV-DIO-hM4Di-mCherry or AAV-DIO-mCherry into the ipsilateral vlPAG. c Representative images of lFN (left) and vlPAG (right) 2 weeks after virus injection. Scale bar, 500 μm (left); 200 μm (right). d Representative image of the vlPAG indicated that few c-Fos expression was co-labelled with EGFP and mCherry double-positive neurons after 3-week continuous i.p. injection of CNO. e Behavioral despair in the FST (left and middle) and anhedonia in the SPT (right) among experimental groups. All mice injected by a rAAV-retro-CaMKIIα -EGFP-Cre vector into the unilateral lFN at the site of wFMNs. Saline + mCherry + CNO − no CRS, mice that received WIM pre-injection of saline, vlPAG injection of AAV-DIO-mCherry, and i.p. injection of CNO without exposure to CRS. Saline + mCherry + CNO − CRS, mice that received WIM pre-injection of saline, vlPAG injection of AAV-DIO-mCherry, and i.p. injection of CNO with exposure to CRS. Saline + hM4Di + CNO − CRS, mice that received WIM pre-injection of saline, vlPAG injection of AAV-DIO-hM4Di-mCherry, and i.p. injection of CNO with exposure to CRS. Saline + hM4Di + Saline − CRS, mice that received WIM pre-injection of saline, vlPAG injection of AAV-DIO-hM4Di-mCherry, and i.p. injection of saline with exposure to CRS. BoNT/A + mCherry + Saline − CRS, mice that received WIM pre-injection of BoNT/A, vlPAG injection of AAV-DIO-mCherry, and i.p. injection of saline with exposure to CRS. n = 8 animals from the group of Saline + mCherry + CNO − no CRS, Saline + mCherry + CNO − CRS and Saline + hM4Di + Saline − CRS, n = 9 animals from the group of Saline + hM4Di + CNO − CRS and BoNT/A + mCherry + Saline − CRS. One-way ANOVA followed by Bonferroni’s multiple comparisons test, F _{(4, 37)} = 11.63, P < 0.0001 for latency to immobility in the FST; F _{(4, 37)} = 10.92, P < 0.0001 for immobility duration in the FST; F _{(4, 37)} = 12.22, P < 0.0001 for sucrose preference in the SPT. The P-value of Bonferroni’s multiple comparisons: Saline + mCherry + CNO − no CRS vs. Saline + mCherry + CNO − CRS: P = 0.0017, Saline + mCherry + CNO − no CRS vs. Saline + hM4Di + Saline–CRS: P = 0.0002, Saline + mCherry + CNO − CRS vs. Saline + hM4Di + CNO − CRS: P = 0.0023, Saline + mCherry + CNO − CRS vs. BoNT/A + mCherry + Saline − CRS: P = 0.0040, Saline + hM4Di + CNO − CRS vs. Saline + hM4Di + Saline − CRS: P = 0.0003, Saline + hM4Di + Saline − CRS vs. BoNT/A + mCherry + Saline − CRS: P = 0.0005 of latency of immobility; Saline + mCherry + CNO − no CRS vs. Saline + mCherry + CNO − CRS: P = 0.0001, Saline + mCherry + CNO − no CRS vs. Saline + hM4Di + Saline − CRS: P = 0.0006, Saline + mCherry + CNO − CRS vs. Saline + hM4Di + CNO − CRS: P = 0.0022, Saline + mCherry + CNO − CRS vs. BoNT/A + mCherry + Saline − CRS: P = 0.0008, Saline + hM4Di + CNO − CRS vs. Saline + hM4Di + Saline–CRS: P = 0.0092, Saline + hM4Di + Saline − CRS vs. BoNT/A + mCherry + Saline–CRS: P = 0.0036 of total time of immobility; Saline + mCherry + CNO − no CRS vs. Saline + mCherry + CNO − CRS: P = 0.0001, Saline + mCherry + CNO − no CRS vs. Saline + hM4Di + Saline − CRS: P < 0.0001, Saline + mCherry + CNO − CRS vs. Saline + hM4Di + CNO − CRS: P = 0.0123, Saline + mCherry + CNO − CRS vs. BoNT/A + mCherry + Saline − CRS: P = 0.0160, Saline + hM4Di + CNO − CRS vs. Saline + hM4Di + Saline − CRS: P = 0.0017, Saline + hM4Di + Saline − CRS vs. BoNT/A + mCherry + Saline − CRS: P = 0.0022 of sucrose preference. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001