Illustration of the caudal periaqueductal gray (cPAG) injection site.

<p>(A) The schematic drawing of Cholera toxin B subunit (CTB) injection. The gray dotted line outlines the border of PAG. Note that to avoid the dye diffusion into superior colliculus (SC), the Hamilton syringe was inclined posteriorly at 30° angle from vertical and advanced 5.2 mm to target the cPAG. (B–E) A coronal section of the injection site. Trace of injection route is shown in (C) and (E). It is evident that CTB did not diffuse into the superior colliculus (SC) and dorsal raphe nuclei (DRN) (C). Large number of 5-HT+ neurons were seen in DRN (green cells in B and D). SC: superior colliculus; IC: inferior colliculus; PAG: periaqueductal gray; DRN: dorsal raphe nuclei; Aq: aqueduct. Scale bars: 300 µm in C (applies to B); 300 µm in E (applies to D).