(A) Viral strategy to express enhanced yellow fluorescent protein (eYFP) or light-sensitive channelrhodopsin (ChR2-eYFP) in synapsin-expressing cells in the dlPAG (dlPAG-syn, left), synapsin-expressing cells in the l/vlPAG (l/vlPAG-syn, middle), and cholecystokinin-expressing cells in the l/vlPAG (l/vlPAG-CCK, right). A fiber optic cannula was then implanted over respective regions. (B) Histology of eYFP expression in dlPAG-syn (left), l/vlPAG-syn (middle), and l/vlPAG-CCK (right). Scale bar, 200 μm. (C) Stimulation protocol in the open field. Blue light (473 nm, 5 ms, 20 Hz) was delivered in alternating 2 min epochs (OFF-ON-OFF-ON) for 8 min total. (D) Diagram indicating center and corners of the open-field assay. (E–G) Example locomotion maps in the open field during laser-on and laser-off epochs of either mice expressing eYFP (top) or ChR2-eYFP (bottom) in dlPAG-syn (E), l/vlPAG-syn (F), and l/vlPAG-CCK (G) populations. (H–M) Bars depict respective behaviors during light-off epochs subtracted from light-on epochs (ON minus OFF). Light delivery to dlPAG of syn-ChR2 mice increased mean speed, jumps, and corner entries compared to dlPAG-syn-eYFP mice (dlPAG-eYFP, n = 5; dlPAG-ChR2, n = 4; unpaired t-tests; speed, *p=0.041; jumps, ***p=0.0005; corner entries, *p=0.044). Light delivery to the l/vlPAG of syn-ChR2 mice reduced mean speed, increased freezing, and reduced corner entries compared to l/vlPAG-syn-eYFP mice (l/vlPAG-syn-eYFP, n = 5; l/vlPAG-syn-ChR2, n = 5; unpaired t-tests; speed, ****p<0.0001; freezing, ****p<0.0001; corner entries, **p=0.0019). Light delivery to the l/vlPAG of CCK-ChR2 mice increased mean speed and corner entries while reducing center time compared to l/vlPAG-CCK-eYFP mice (l/vlPAG-CCK-eYFP, n = 17; l/vlPAG-CCK-ChR2, n = 14; unpaired t-tests; speed, ***p=0.001; corner entries, ****p<0.0001; center time, **p=0.0023). Importantly, time spent in corners increased during light delivery to l/vlPAG-CCK-ChR2 mice compared to l/vlPAG-CCK-eYFP mice (unpaired t-test, **p=0.006). Errorbars: mean ± SEM.