Immunohistochemical characterization of the intrinsic cardiac neural plexus in whole-mount mouse heart preparations

Abstract

Background: The intrinsic neural plexus of the mouse heart has not been adequately investigated despite the extensive use of this species in experimental cardiology.

Objective: The purpose of this study was to determine the distribution of cholinergic, adrenergic, and sensory neural components in whole-mount mouse heart preparations using double immunohistochemical labeling.

Methods/results: Intrinsic neurons were concentrated within 19 ± 3 ganglia (n = 20 mice) of varying size, scattered on the medial side of the inferior caval (caudal) vein on the right atrium and close to the pulmonary veins on the left atrium. Of a total of 1,082 ± 160 neurons, most somata (83%) were choline acetyltransferase (ChAT) immunoreactive, whereas 4% were tyrosine hydroxylase (TH) immunoreactive; 14% of ganglionic cells were biphenotypic for ChAT and TH. The most intense ChAT staining was observed in axonal varicosities. ChAT was evident in nerve fibers interconnecting intrinsic ganglia. Both ChAT and TH immunoreactivity were abundant within the nerves accessing the heart. However, epicardial TH-immunoreactive nerve fibers were predominant on the dorsal and ventral left atrium, whereas most ChAT-positive axons proceeded on the heart base toward the large intrinsic ganglia and on the epicardium of the root of the right cranial vein. Substance P-positive and calcitonin gene-related peptide-immunoreactive nerve fibers were abundant on the epicardium and within ganglia adjacent to the heart hilum. Small intensely fluorescent cells were grouped into clusters of 3 to 8 and were dispersed within large ganglia or separately on the atrial and ventricular walls.

Conclusion: Although some nerves and neuronal bundles of the mouse epicardial plexus are mixed, most express either adrenergic or cholinergic markers. Therefore, selective stimulation and/or ablation of the functionally distinct intrinsic neural pathways should allow the study of specific effects on cardiac function.

Fig. 1

Whole-mount preparation. Both atrial appendages, pulmonary vein roots, and the major part of the ventricles were dissected in order to flatten the atria. ChAT-IR or cholinergic neural structures are labeled red, TH-IR or sympathetic structures are green. Arrows indicate the extrinsic nerves accessing the mouse heart, whereas arrowheads point to intrinsic ganglia. The boxed areas in panel a are enlarged in the insets to illustrate: (b) the predominance of ChAT-IR ganglionic cells; (c) the small ganglia, and single neurons; (d) the nerves accessing the heart on the anterior side of the left cranial vein root in which the TH-IR nerve fibers predominate compared to neighboring solitary ChAT-IR nerve fibers; (e) the nerves accessing the heart on the medial side of the right cranial vein root with absolute predominance of TH-IR nerve fibers; (f) the considerable difference in density of the cholinergic neural meshwork on the right cranial vein root (RCV) compared with the rest of right atrial wall; (f) the occurrence of ChAT-IR fibers together with TH-IR fibers in epicardial nerves. Abbreviations: PT - orifice of pulmonary trunk, AO – orifice of aorta, RCV – orifice of right cranial vein, CV – orifice of caudal vein, LCV - orifice of left cranial vein, PVs – orifice of left ant middle pulmonary veins, RPV – orifice of right pulmonary vein.

Fig. 2

The number of neurons plotted against the area of 132 ganglia from 15 mouse hearts. The straight line indicates the linear regression of the plotted data. Each point corresponds to one of the analyzed ganglia (some points overlap in the graph). R – correlation coefficient at P < 0.0001.

Fig. 3

a–c: Microphotographs illustrating the predominance of ChAT-IR ganglionic cells inside a ganglion that contains also the biphenotypic (arrowheads) and small intensively fluorescent (SIF) (arrows) cells immunoreactive to TH. Note that immunostaining for ChAT (b) does not exhibit entthe SIF cells (arrows), while only the double-channel fluorescence of simultaneous immunostainings for TH and ChAT (c) reveals reliably the biphenotypic neurons (arrowheads) that were dimly seen in mono-channel fluorescence applied to display immunostaining for TH (a); d–f: Microphotographs demonstrating three types of neurons in small ganglion of the mouse heart identified applying mono-channel (d and e) and double-channel (f) fluorescence. Note the baskets of ChAT-IR neural terminals that surround the neurons and contain numerous varicosities. White crosses indicate the neuron that is exclusively positive to TH, asterisks - neurons positive only to ChAT, and diamonds - two biphenotypic nerve cells; g–i: Microphotographs confirming the neuronal origin of all ganglionic cells and showing the diversity of sizes and staining intensities of TH-IR neurons located within one ganglion. Note the intensely (diamonds) and weakly (crosses) stained TH-IR neurons, which are positive for the general neuronal marker PGP 9.5.

Fig. 4

Whole-mount preparation of the interatrial (a–c) and interventricular (d–f) septa illustrating the pathways of intrinsic nerves proceeding toward the atrioventricular node region (a) as well as along the His bundle and its right branch (d). Note the predominance of ChAT-IR fibers in the region of the atrioventricular node, at His bundle and it right branch. The nerves (arrowheads) from epicardial ganglia (solid white arrow) extend toward the atrioventricular node and form the dense neural meshwork therein. The boxed areas in panel a are enlarged as the insets in b and c, while ones in panel d – as the insets in e and f. Double arrowheads in a and b point out the solitary ChAT-IR neuron located beneath the endocardium of the interatrial septum. Asterisks in d track the blood vessels in the interventricular septum. Abbreviations: FO – fossa ovalis, AVN - atrioventricular node region, CS – orifice of coronary sinus.

Fig. 5

a–c: Microphotographs showing the myocardial preterminal and terminal aspects of the ChAT-IR and TH-IR axons that contain numerous varicosities and spine-like processes. Arrowheads indicate the ChAT-IR axons, while arrows - the axons with a mixture of TH and ChAT; d-i: Microphotographs illustrating SP-IR nerve fibers in close vicinity to atrial myocytes (d–f) and CGRP-IR nerve fibers between the ChAT positive ganglionic cells (g–i). Note the numerous varicosities and button-like terminal endings of the CGRP-IR nerve fibers at ChAT-IR nerve cells. In d–f panels, asterisks point to two atrial myocytes, in which myofibril striations are seen, while in g–i panels, double arrowheads – the varicosities of CGRP-IR nerve fiber.