Figure 1. Cholecystokinin-expressing (CCK+) cells comprise approximately 5% of lateral/ventrolateral periaqueductal gray (l/vlPAG) neurons and are primarily glutamatergic.

(A) Example histology images showing immunostaining of pan-neuronal marker NeuN (top row), viral-mediated expression of GFP in CCK-expressing cells (middle row), and overlay of NeuN and CCK-GFP (bottom row) in the dorsolateral (left column), lateral (middle column), and ventrolateral (right column) PAG. Scale bars, 10 μm. (B) Raw counts of CCK-GFP+ and NeuN+ cells in the dorsolateral PAG (dlPAG), lPAG, and vlPAG. (C) Fraction of NeuN-labeled cells that are also GFP-labeled in the dlPAG and l/vlPAG. CCK-expressing cells comprise ~5% of l/vlPAG neurons and constitute significantly more of l/vlPAG neurons than dlPAG neurons (n = 4; paired t-test, **P=.0032). (D) Immunostaining of glutamatergic marker vGlut2 in CCK cells. Example histology images showing vGlut2 (top), CCK-GFP (middle) and vGlut2/GFP overlay (bottom). White arrow indicates vGlut2+/GFP+ cell. Dashed outline indicates vGlut2+/GFP- cell. Scale bar, 10 μm. (E) 9.6% of vGlut2-labeled cells in the l/vlPAG are also GFP-labeled (n = 4; 302 vGlut2/GFP+ of 3115 vGlut2+ cells). (F) A majority (94.8%) of GFP-labeled cells in the l/vlPAG are also vGlut2-labeled (n = 4; 302 vGlut2+/GFP+ of 317 GFP+ cells). Errorbars: mean ± SEM.

Figure 1—figure supplement 1. In situ hybridization of vglut2 and cholecystokinin-expressing (CCK) cells in the lateral/ventrolateral periaqueductal gray (l/vlPAG) shows double labeling of vglut2 and CCK.

(A) In situ hybridization labeling of l/vlPAG neurons showing mRNAs for CCK (green) and VGLUT2 (red). Nuclei stained by DAPI are shown in blue. Example image (×40 objective) of vglut2-labeled cells (red rectangle) and double-labeled vglut2/CCK-labeled (green rectangle) in l/vlPAG. Scale bar, 20 µm. (B) Zoomed-in images of subregions in (A). Scale bar, 20 µm. (C) 8.58% of vglut2-labeled cells are also CCK-labeled (15 of 183 total cells). Errorbars: mean ± SEM.