3D single cell scale anatomical map of sex-dependent variability of the rat intrinsic cardiac nervous system

Figures

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  Figure 1. Spatially tracked anatomical map of rat ICNS to enable comparison of variability within and across sexes (A) Schematic representation of the workflow to acquire male and female rat ICNS maps, analysis of the data using cluster analysis of distinct groups of neurons, and alignment of data sets onto a generic scaffold. (B) Posterior whole heart views of four male and three female rat hearts. Yellow: males; Pink: females.

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  Figure 2. Quantity and distribution of neurons within the rat ICNS across individuals and sexes (A and B) The anatomical map of rat ICNS across four males (A) and three females (B). Each dot represents a single neuron. The anatomical orientation corresponding to the whole heart is indicated. Blue: males; Purple: females. (C) Quantitative analysis of the total number of neurons mapped and annotated within seven rat hearts.

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  Figure 3. ICNS neurons are consistently localized around the left atrium (A) Partitioning around medoids (PAM) clustering analysis in a male rat heart delimited 12 groups of ICNS neurons found at the base of the heart, visualized here with each color representing a distinct cluster of cells. (B) Histological sections throughout various levels of the image stack corresponding to the male rat heart shown in panel (A) were evaluated to identify which clusters were distributed around the left atrium. (C) In addition to the histological context, clusters near the left atrium were visualized in their 3D spatial context using Neurolucida Explorer as an independent analysis. (D) The PAM-identified clusters were re-colorized as dark blue and gray to represent ICNS neurons proximal and distal to the left atrium, respectively. (E–J) 3D spatial evaluation for the remaining male (E-G) and female (H-J) rat hearts identified the neurons that are proximal and distal to the left atrium. The neurons proximal to the left atrium are re-colorized as dark blue (male) and purple (female), whereas the neurons distal to the left atrium are visualized in gray. Scale bar: 250 μm.

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  Figure 4. Data-driven analysis of ICNS demonstrates preferential organization of ICNS neurons in select cardiac anatomical regions (A–C) ICNS neurons from two male rat hearts that organize around the pulmonary veins (PV) (A), the root of the superior vena cava and right atrium (SVC-RA) (B), and the atrioventricular sulcus (C). (A) ICNS neurons encircling the pulmonary veins highlighted in green. (B) ICNS neurons around the root of the superior vena cava and right atrium (SVC-RA) are highlighted in yellow. (C) Clusters of ICNS neurons present at the left atrioventricular sulcus near the coronary sinus are highlighted in pink. In (A-C), all neurons not emphasized as being associated with the particular region of interest are shown in gray.

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  Figure 5. ICNS neurons are localized along the interatrial sulcus (A–G) Partial projections and histology for M54-5 (A,C,E) and M54-8 (B,D,F). (A,B) A superior view of partial projections of a selective thickness of heart tissue visualized as 3D image volumes for a pair of male rat hearts. Overlaying contours of the left and right atria onto the ICNS highlights a large number of ICNS neurons distributed along the anterior interatrial sulcus. (C,D) A left superior view of the posterior aspect of the partial projections illustrates the distribution of ICNS neurons along the anterior interatrial sulcus that are obscured by the right pulmonary artery. (E,F) Cardiac histology shows the location of specific clusters in relation to the left and right atria as well as the right pulmonary artery. Scale bar: 250 μm. (G) Such a pattern of localization to these cardiac anatomical regions was observed across all the male and female rat hearts. The colors green, yellow, and pink correspond to ICNS neurons around the PV, the root of the SVC-RA, and the left atrioventricular sulcus, respectively (From Figure 4).

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  Figure 6. Mapping individual rat ICNS and cardiac anatomy onto a generalized 3D scaffold for comparison across animals (A) Workflow for registering individual ICNS into a 3D scaffold, fitting the scaffold to the individual cardiac anatomical features, and projection of individual cells on the scaffold to embed the ICNS in its elements. (B) An overview of the fitting and mapping processes from the original 3D anatomical segmented data of one of the male rat hearts viewed from two different angles. The original contour data, fitted scaffold and generic scaffold are shown. (C) The heart is visualized from a left superior angle to appreciate how the neuron locations in the original data are projected onto the generic scaffold. Yellow spheres represent positions of the original ICNS locations prior to transforming the data onto the fitted scaffold; orange spheres represent the new ICNS locations embedded in the scaffold. (D) Integration of two data sets onto one generic scaffold. (E) Looking at the same regions of interest in Figures 4 and 5, varying colors of pink are used to indicate the proportion of neuronal overlap between the two male datasets from panel (D). Additional scaffolds showing…

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  Figure 7. Comparison of male and female ICNS (A and B) Whole heart posterior view of PAM clustered neurons of male (A) and female (B) ICNS. (C and D) Flatmap projections derived from PCA plots are used to show the spatial data and packing density of the neurons in male (C) and female (D) ICNS colored for PAM-identified clusters (Flatmaps for all hearts can be found in Figure S1, albeit not colored by PAM cluster). (E and F) Histological tissue sections containing PAM colored neurons in relation to the four regions of interest are shown in a 2D context to support the 3D visual data for male (E) and female (F) ICNS. Scale bar: 250 μm.