Catecholaminergic and Cholinergic Axon Distribution and Morphology in Whole Flat-Mount Mouse Atria

The autonomic nervous system, comprising sympathetic and parasympathetic divisions, plays a pivotal role in regulating cardiac functions. Despite its critical importance, our understanding of the distribution, morphology, and interactions between cardiac sympathetic and parasympathetic axons remains limited. To address this gap, we prepared flat-mounts of the whole of atria of C57BL/6 mice and employed single and dual immunohistochemical labeling techniques to determine tyrosine hydroxylase (TH) immunoreactive (IR) axons, a marker for sympathetic innervation, and vesicular acetylcholine transporter (VAChT) IR axons, a marker for parasympathetic innervation. Our results were: In single TH-labeling: 1) Four to five major extrinsic TH-IR nerve bundles entered the right (RA) and left atria (LA) via the superior vena cava and left precaval vein. 2) Large TH-IR axon bundles branched into smaller bundles that eventually formed terminals in all atrial regions, with the sinoatrial (SA) node region showing the highest density. 3) Many cardiac principal neurons (PNs) within intrinsic cardiac ganglia (ICG) and small intensely fluorescent cells were strongly TH-IR. 4) TH-IR varicose axons travelled along blood vessels and around adipose tissues. In single VAChT-labeling: 1) In contrast to TH, no large VAChT-IR bundles were found. However, VAChT-IR varicose axons were densely distributed throughout the left and right atria with highest density located in the SA node. 2) VAChT weakly labeled PNs within all ICG on the dorsal surfaces of the LA and RA. 3) VAChT strongly labeled terminals (vagal efferent) that tightly encircled individual PNs of the ICG. 4) Some VAChT-IR terminals were also observed along the blood vessels and around adipose tissues. In dual-labeling: 1) TH-IR and VAChT-IR axons often diverged from epicardial bundles and traveled in parallel and in close proximity (less than 1 μm apart) before forming terminal structures in the epicardium and myocardium. 2) These axons interdigitated, featuring alternating varicosities along their lengths in close juxtaposition. 3) TH+VAChT-IR PNs (15-30%) within ICG formed terminal structures in the cardiac muscles without projecting to other PNs, but they formed varicose axons in the cardiac muscles. 4) Individual neurons in the ICG are tightly encircled by VAChT-IR varicosities while TH-IR axons passed through the ICG without making varicose contacts with PNs. 5) Both TH-IR and VAChT-IR axons ran along the atrial vasculature and around fat pads This study provides insights into the structural organization of sympathetic and parasympathetic innervation in the heart and their potential interaction under the anatomical context. These findings are also essential for advancing our understanding of autonomic remodeling in cardiovascular diseases. Supported by NIH 1 U01 NS113867-01 (ZC), NIH R15 1R15HL137143-01A1 (ZC), 2R01HL137832-05 (YL). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.