Fig. 7: Chemogenetic or optogenetic activation of PMC-projecting l/vlPAG SST⁺ neurons promotes urination in freely moving mice.

a Schematic for AAV-Retro-FLEX-Flpo injection into the PMC and AAV-fDIO-hM3D-mCherry injection into the l/vlPAG of SST-Cre mice (left). Representative image of hM3D-expressing somas in the l/vlPAG (right). b Experimental timeline for investigating the effects of CNO-induced activation on urination. c Representative urine spots deposited on filter papers from hM3D-expressing mice treated with saline or CNO. d Raster plots showing urination events during the 4-day behavioral test. e Daily quantification of urination events on filter paper from hM3D-expressing mice (n = 8 mice). ***P < 0.001 (Friedman’ s test); day 1 vs. day 2, *P = 0.016; day 3 vs. day 4, **P = 0.008. f Quantification of the urination interval (left) and total urine area (right) following saline or CNO administration from hM3D-expressing mice (n = 8 mice). Left, **P = 0.002 (saline versus CNO, paired t test); right, *P = 0.012 (saline versus CNO, Wilcoxon signed-rank test). g Schematic of AAV-Retro-FLEX-Flpo injection into the PMC and AAV-fDIO-ChR2-mCherry injection into the l/vlPAG of SST-Cre mice (left) and optical fiber implantation above the l/vlPAG four weeks following viral expression (right). Representative images of ChR2 expression in PMC-projecting l/vlPAG SST⁺ neurons (right). h Schematic of optogenetic activation experiment and real-time tracking of urination events in freely moving mice. i Raster plot of urination duration (yellow bars) in each trial during and after photostimulation of PMC-projecting l/vlPAG SST⁺ neurons that express ChR2 (n = 120 trials from 8 mice) or mCherry (n = 105 trials from 7 mice). White bars indicate periods without urination and blue bars indicate light-stimulation periods. j Quantification of the percentage of photostimulation trials inducing urination (left). n = 8 mice for ChR2 group, n = 7 mice for mCherry group. **P = 0.001 (ChR2 group versus mCherry group, Wilcoxon rank-sum test). Summary of the latency of light-evoked urination following photostimulation of ChR2-expressing PMC-projecting l/vlPAG SST⁺ neurons (right). All data are presented as mean ± SEM.