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. 2022 Jun 8;11:e77115. doi: 10.7554/eLife.77115

Figure 2. Lateral/ventrolateral periaqueductal gray-cholecystokinin-expressing (l/vlPAG-CCK) cell stimulation induced a repertoire of behaviors distinct from pan-neuronal l/vlPAG and dorsolateral PAG (dlPAG) activation.

(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.

Figure 2.

Figure 2—figure supplement 1. ChR2 expression and fiber placement in the periaqueductal gray (PAG) in coronal brain sections.

Figure 2—figure supplement 1.

ChR2 expression is depicted in shading and fiber placement is represented by dots for (A) synapsin-expressing cells in the dorsolateral PAG (dlPAG-syn) mice, (B) synapsin-expressing cells in the lateral/ventrolateral PAG (l/vlPAG-syn) mice, and (C) cholecystokinin-expressing cells in the l/vlPAG (l/vlPAG-CCK) mice. Fibers targeting the l/vlPAG in (B) and (C) were placed at 15° angle. All fiber placements were unilateral and counterbalanced across left and right PAG. Center of fiber was determined at the widest point in histological slices.