Topographical Mapping of Catecholaminergic Axon Distribution and Morphology in the Flat‐Mounts of the Mouse Heart

Abstract

The sympathetic nervous system plays a crucial role in the control of heart functions. However, the distribution and morphology of sympathetic postganglionic innervation of the heart has not been well elucidated, which has significantly impeded the understanding of the sympathetic neuroanatomical organization and control of the heart. Our goal is to provide a map of the topographical innervation of sympathetic postganglionic neurons in the whole heart of mice. In this study, we used Tyrosine hydroxylase (TH) as a marker for sympathetic axons and applied a combination of state‐of‐the‐art techniques, including confocal microscopy, Zeiss Imager microscopy, flat‐mount tissue processing and immunohistochemistry, Neurolucida 360 Tracing and integration of the tracing data onto a 3D heart scaffold. We found that 1) Multiple large extrinsic TH‐IR nerve bundles entered the heart and branched out into smaller axons which covered the whole left and right atria and ventricles, 2) These TH‐IR bundles branched out into smaller axon bundles and ramified into individual axons covering the whole left and right atria and ventricles. 3) The TH‐IR axons formed an extensive neural network in the epicardium and axons formed varicose synaptic endings within the cardiac muscle. 4) TH‐IR axons and terminals were densest in the sinoatrial (SA) and atrioventricular (AV) node regions. 5) TH‐IR axons innervated blood vessels and fat cells. 6) Many principal neurons in intrinsic cardiac ganglia and small intensely fluorescent (SIF) cells were TH‐IR. 7) However, TH‐IR axons do not appear to innervate any cardiac principal neurons and SIF cells. 8) TH‐IR axon innervation will be integrated into a 3D scaffold. Our work will provide a comprehensive topographical map of the catecholaminergic efferent axon distribution, innervation, and morphology of the whole heart at single cell/axon/synapse scale that will be used to create a cardiac sympathetic‐brain atlas. It will also provide an anatomical reference to evaluate the altered sympathetic cardiac control in pathological conditions. The first two authors contributed equally to this work.