Catecholaminergic axon innervation and morphology in flat‐mounts of atria and ventricles of mice

Abstract

Sympathetic efferent axons regulate cardiac functions. However, the topographical distribution and morphology of cardiac sympathetic efferent axons remain insufficiently characterized due to the technical challenges involved in immunohistochemical labeling of the thick walls of the whole heart. In this study, flat-mounts of the left and right atria and ventricles of FVB mice were immunolabeled for tyrosine hydroxylase (TH), a marker of sympathetic nerves. Atrial and ventricular flat-mounts were scanned using a confocal microscope to construct montages. We found (1) In the atria: A few large TH-immunoreactive (IR) axon bundles entered both atria, branched into small bundles and then single axons that eventually formed very dense terminal networks in the epicardium, myocardium and inlet regions of great vessels to the atria. Varicose TH-IR axons formed close contact with cardiomyocytes, vessels, and adipocytes.Multiple intrinsic cardiac ganglia (ICG) were identified in the epicardium of both atria, and a subpopulation of the neurons in the ICG were TH-IR. Most TH-IR axons in bundles traveled through ICG before forming dense varicose terminal networks in cardiomyocytes. We did not observe varicose TH-IR terminals encircling ICG neurons. (2) In the left and right ventricles and interventricular septum: TH-IR axons formed dense terminal networks in the epicardium, myocardium, and vasculature. Collectively, TH labeling is achievable in flat-mounts of thick cardiac walls, enabling detailed mapping of catecholaminergic axons and terminal structures in the whole heart at single-cell/axon/varicosity scale. This approach provides a foundation for future quantification of the topographical organization of the cardiac sympathetic innervation in different pathological conditions.

Chronic intermittent hypoxia remodels catecholaminergic nerve innervation in mouse atria

“…11 and ), ICG neurons were clustered together following CIH compared to RA. Similar to our recent study (Bizanti et al., 2023; Zhang et al., 2023), and in contrast to vagal efferent axons, TH‐IR axons were seen to travel through TH‐IR neurons in the ganglia rather than tightly encircle individual neurons. TH‐IR axons coursing through ICG were observed in the optical sections (Fig.…”

“…Consistent with our previous studies (Bizanti et al., 2023), we observed that TH‐IR bundles and axons primarily travelled through the ICG without encircling individual ICG principal neurons as seen by parasympathetic axons in RA after CIH (Cheng, 2017). The consistent distribution of ICG was evident, with most ganglia noted in the left atrial region.…”

“…The heart, lung and trachea were removed from the chest and postfixed overnight in Zamboni's fixative at 4°C. The heart was placed and pinned into a dissecting dish containing PBS (pH 7.4) and the specimen was further dissected using a surgical microscope (Leica, Wetzlar, Germany), as described previously (Ai et al., 2007; Bizanti et al., 2023; Cheng et al., 1997; Lin et al., 2008). In brief, the lungs and trachea were removed with fine forceps, the atria were separated from the ventricles, and the aortic arch was removed from the atria.…”

“…Tissue processing and immunolabelling were performed as described previously (Bizanti et al, 2023;Zhang et al, 2023;Li et al, 2014). Following dissection, the tissues were washed six times for 5 min in 0.1M PBS (pH 7.4), then immersed for 48 h in a blocking reagent (2% bovine serum albumin, 10% normal donkey serum, 2% Triton X-100, 0.008% NaN 3 in 0.1 M PBS, pH 7.4).…”

Topographical mapping of catecholaminergic axon innervation in the flat-mounts of the mouse atria: a quantitative analysis

“…Hearts along with the lungs and trachea were removed from the chest and postfixed overnight in 4% paraformaldehyde at 4 °C. The heart was placed and pinned into a dissecting dish lined with Sylgard and containing PBS (0.1 M, pH = 7.4), and the specimen was further dissected using a Leica Stereo microscope as described previously 25,27,28,32,33 . To reveal the intact network of sympathetic postganglionic atrial innervation, we removed the heart from the surrounding tissues (lungs, aortic arch and trachea).…”