Problem
Spinal afferents, nerves that send signals from the peripheral organs to the spinal cord, are crucial for detecting and transmitting pain signals. A clear understanding of their distribution and morphology in the heart is essential due to their role in cardiac sensory innervation and remodeling in cardiovascular diseases, such as angina pectoris and chronic heart failure. However, detailed mapping of spinal afferent innervation in the heart remains challenging, largely due to limitation in tracer selection, the thickness of heart tissue, and the difficulty of accessing the upper thoracic dorsal root ganglia (DRGs) for selective spinal afferent labeling.
Previous studies have mapped peptidergic axons in mouse atria using immunohistochemical labeling of markers such as calcitonin gene-related peptide and substance P (Li et al., 2014, Chen et al., 2024). Although these markers label spinal afferent axons from the DRG, they may also label vagal afferents from the nodose ganglia, potentially from both sides. Additionally, studies utilizing transgenic mice that overexpressed Vesicular Glutamate Transporter Type 2 in the primary afferents could not differentiate its specific origins (Sahoglu et al., 2023). Consequently, the distribution and morphology of spinal afferents and their terminals within the entire atria and ventricles remains a vital question, yet to be answered.
Solution
To address these issues, we employed an anterograde tracing technique that utilizes whole flat-mount tissue to specifically target spinal afferent nerve endings in the rat atria, effectively avoiding labeling of other extrinsic afferent and efferent nerve types (Figure 1). Additionally, single spinal afferent axons were traced, digitized and analyzed in their three-dimensional space using the MBF Neurolucida system.This approach can selectively visualize the detailed structure and precise location of spinal afferent endings without labeling other nerve classes that also innervate the atria.
Figure 1: Schematic illustration of anterograde tracer dextran biotin (DB) injection into the dorsal root ganglion (DRG). After exposing the C8-T3 vertebrae, the intervertebral foramina located between two consecutive thoracic vertebral spinous processes were carefully drilled. The tracer (greenish-blue) was injected into the DRG.
Figure 2: Anterograde labeling of a single spinal afferent axon with extensive branch arborization in the left atrium. (a) Contour of a representative left atrium and the location of a single axon within the atrium. (a’) Neurolucida tracing of the axon. The parent axon (PA) is indicated with a black arrow. (a’1) A rotational view of the Neurolucida tracing in (a’) (a’1; counterclockwise y: 800). The red circles in (a’) and (a’1) mark the first bifurcation point of this PA. Landmarks, including red arrowheads, red arrows and red circles, designate the exact corresponding positions of the tracing from different angles in (a’) and (a’1), respectively. (b,c,d) Partial projections corresponding to the same letters in (a’) showed the parent axon near epicardial adipose tissue (b) and its varicose branches innervating the cardiac muscle (c and d). Free terminals are observed in (d) (black arrows), and in (a’) and (a’1) (red arrowheads). AT, adipose tissue; CM, cardiac muscle; Epi, epicardium; Myo, myocardium; LAu, left-atrium auricle; PV, pulmonary vein.
Figure 3: Distribution of spinal afferent axons in the flat-mount of whole atria (LA: n=7, RA: n=8). In the reconstructed connected left and right atria (top), the dots represent the first bifurcation of the axon digitized using the Neurolucida system. The axons covered the entire flat-mount preparations of both left and right atria, with a predominant distribution on the left. The axons from all samples were overlaid on a common atrial contour using a landmark-based transformation tool (bottom). LAu, left atrium auricle; PV, pulmonary veins; RAu, right atrium auricle; LPCV, left pre-caval vein; SVC, superior vena cava; IVC, inferior vena cava.
Impact
This study presents an unprecedented detailed mapping of cardiac spinal afferent axon distribution in the atrial wall at the single cell/axon/varicosity scale. The precise tracing of these axons paves the way for future quantitative assessment of the spinal afferent receptive field and terminal structures within different cardiac layers and targets. Currently, Dr. Cheng’s team is adapting these techniques to determine the spinal afferent axon distribution in the thick walls of whole ventricles. This work emerges as a novel approach to building an anatomical basis for future research of functional roles and remodeling of cardiac spinal afferent in disease models.
Figure 4: Schematic representation of terminal structures and targets of spinal and vagal afferents in different layers of the rat atrium. Both spinal and vagal afferents project to all three layers: epicardium (Epi), myocardium (Myo), and endocardium (Endo). In the Epi, spinal afferents make some close contacts with cardiac ganglionic neurons and adipose tissue, whereas vagal afferents selectively form pericellular terminal structures around small intensely fluorescent (SIF) cells without innervating any cardiac ganglionic neurons. In the Myo, spinal afferents display a range of axonal structures, from simple to complex branching patterns, whereas vagal afferents form specialized intramuscular endings. In the Endo, some spinal afferents branch into free nerve endings, whereas vagal afferents form many distinct terminal “flower-sprays” and end-nets.
