Fig. 1. ICNs exhibit the cholinergic phenotype.

(A) Low magnification epifluorescence image of atrial section stained for the pan-neuronal marker PGP 9.5. Arrow indicates the location of a ganglion shown at higher magnification in confocal images (B) and (C). PGP 9.5-IR neuronal cell bodies (A, B) also labeled for the cholinergic marker ChAT (C). More intense ChAT staining was evident in preganglionic varicosities that surrounded many of these neurons (C, insert). (D–F) Confocal images of another section, doubled labeled for CHT (D) and VAChT (E), showed that these cholinergic markers were colocalized (yellow in the overlay, panel F) in varicosities around ICNs and in many atrial nerve fibers (open arrows). Inserts at lower left show boxed regions at higher magnification. Panels (B) and (C) show single optical sections, and panels (D–F) show maximum projection images compiled from an 8 µm confocal series. a – aorta, ra – right atrium, and rv – right ventricle. Scale bars indicate 500 µm in (A) and 100 µm in (B and C) and (D–F).