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Molecularly defined circuits for cardiovascular and cardiopulmonary control

Nature volume 606, pages 739–746 (2022)Cite this article

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Abstract

The sympathetic and parasympathetic nervous systems regulate the activities of internal organs1, but the molecular and functional diversity of their constituent neurons and circuits remains largely unknown. Here we use retrograde neuronal tracing, single-cell RNA sequencing, optogenetics and physiological experiments to dissect the cardiac parasympathetic control circuit in mice. We show that cardiac-innervating neurons in the brainstem nucleus ambiguus (Amb) are comprised of two molecularly, anatomically and functionally distinct subtypes. The first, which we call ambiguus cardiovascular (ACV) neurons (approximately 35 neurons per Amb), define the classical cardiac parasympathetic circuit. They selectively innervate a subset of cardiac parasympathetic ganglion neurons and mediate the baroreceptor reflex, slowing heart rate and atrioventricular node conduction in response to increased blood pressure. The other, ambiguus cardiopulmonary (ACP) neurons (approximately 15 neurons per Amb) innervate cardiac ganglion neurons intermingled with and functionally indistinguishable from those innervated by ACV neurons. ACP neurons also innervate most or all lung parasympathetic ganglion neurons—clonal labelling shows that individual ACP neurons innervate both organs. ACP neurons mediate the dive reflex, the simultaneous bradycardia and bronchoconstriction that follows water immersion. Thus, parasympathetic control of the heart is organized into two parallel circuits, one that selectively controls cardiac function (ACV circuit) and another that coordinates cardiac and pulmonary function (ACP circuit). This new understanding of cardiac control has implications for treating cardiac and pulmonary diseases and for elucidating the control and coordination circuits of other organs.

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Fig. 1: scRNA-seq of AmbCardiac neurons identifies a genetic signature of brainstem parasympathetic neurons.
Fig. 2: Two molecularly and anatomically distinct types of AmbCardiac neurons.
Fig. 3: Cardiac ganglion innervation patterns of ACP and ACV neurons.
Fig. 4: Cardiac effects of optogenetic activation of left and right ACP and ACV neurons.
Fig. 5: Distinct ACP and ACV activation patterns in baroreceptor and dive reflexes.
Fig. 6: ACP neurons innervate the lung and mediate bronchoconstriction.
Fig. 7: Parallel cardiovascular and cardiopulmonary control circuits.

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Data availability

scRNA-seq data from AmbCardiac and AmbLaryngeal neurons are available at the Gene Expression Omnibus under accession number GSE198709. Source data are provided with this paper.

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Acknowledgements

We thank B. Hsueh for advice on optogenetics and assistance with pilot experiments; K. Yackle for guidance on neuron aspiration for scRNA-seq; J. Zigman for providing the Ghsr-IRES-cre mouse line; K. Ritola and the Janelia Viral Tools facility for producing the bReaChES vector; A. Olson and the Stanford Neuroscience Microscopy Service (supported by NIH NS069375) for providing an electrophysiology rig; the Stanford Functional Genomics Facility for providing sequencing services; the Stanford Research Computing Center for providing computational resources through the Sherlock cluster; K. Travaglini for discussions on scRNA-seq analysis; S. Chang for discussions on ECG traces; and the Krasnow laboratory for helpful discussions and comments on the manuscript. A.V. was supported by the Stanford Medical Scientist Training Program and a Lubert Stryer Bio-X Stanford Interdisciplinary Graduate Fellowship, A.R.Y. was supported by a Walter V. and Idun Berry Fellowship, and Y.L. was supported by a Damon Runyon Fellowship. M.A.K. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Biochemistry, Wall Center for Pulmonary Vascular Disease, and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA

    Avin Veerakumar, Andrea R. Yung, Yin Liu & Mark A. Krasnow

  2. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Avin Veerakumar

  3. Medical Scientist Training Program, Stanford University School of Medicine, Stanford, CA, USA

    Avin Veerakumar

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Contributions

A.V. and M.A.K. conceived the project. A.V. performed experiments. A.R.Y. performed lung projection mapping. A.R.Y. and A.V. performed dual lung mechanics and ECG recording experiments. Y.L. found calbindin-positive terminals in lung ganglia. A.V., A.R.Y. and M.A.K analysed and interpreted data. A.V. and M.A.K. wrote the manuscript with input from all authors.

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Correspondence to Mark A. Krasnow.

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Extended data figures and tables

Extended Data Fig. 1 Distribution of cardiac-innervating neurons in the nucleus ambiguus (Amb).

a, Sagittal schematic view displaying locations of Amb neurons (circles) within a postnatal day 2 Amb nucleus. Map is an overlay showing the locations of Amb neurons from all sagittal sections spanning a single Amb nucleus. Green fill circles, cardiac Amb neurons retrograde labeled by cholera toxin B (CTB) injection into pericardial space (CTB>Heart). Grey fill circles, other Amb neurons. Note cardiac Amb neurons localize primarily to “external formation” of Amb, surrounding the principal rostral-caudal column of Amb neurons. b, Representative sagittal section of Amb with AmbCardiac neurons labeled in green by retrograde labeling by CTB injection in heart (CTB>Heart). Retrograde labeled cells localized to the Amb external formation. AmbC, nucleus ambiguus compact formation. nVII, facial motor nucleus. Bar, 100 µm.

Extended Data Fig. 2 Quality control data for Amb single cell RNAseq.

a, Aligned reads per cell in combined AmbCardiac and AmbLaryngeal dataset. Cells with less than 500,000 reads were excluded from analysis. b, Genes detected per cell. c, Percent of reads per cell that aligned to ERCC spike-in RNA. d, Pan-neuronal genes Nefl, Tubb3, Snap25, and cholinergic gene Slc18a3 were highly expressed across all clusters from Fig. 1b. e, Glial genes were expressed at similarly low levels across the 3 clusters from Fig. 1b, indicating they did not contribute to clustering. Gja1; astrocyte marker, C1qc; microglia marker, Pdgfra; oligodendrocyte precursor cell marker, Opalin; oligodendrocyte marker. f, Examples of marker genes that significantly contributed to clustering of the 3 neuron types (Tbx3; AmbCardiac marker, Pappa2; ACP marker, Hoxa5; ACV marker, Calca; AmbLaryngeal marker).

Source data

Extended Data Fig. 3 AmbCardiac markers are expressed in other brainstem parasympathetic nuclei.

In situ hybridization data (from Allen Brain Atlas15) showing expression (dark purple) of the indicated AmbCardiac-specific genes (Celf6, Kcna5, Tbx3, Dgkb, from Fig. 1) in parasympathetic neurons across the brainstem. Top row, Amb; middle row, dorsal motor nucleus of the vagus (10N); bottom row, lacrimal and salivatory nuclei (Lac/Sal). Note the genes are expressed in a subset of Amb neurons (top row panels), as predicted by scRNAseq (Fig. 1), but also in parasympathetic neurons of the dorsal motor nucleus of vagus (10N) that innervate thoracic and abdominal viscera (second row panels). Note absence of expression in the adjacent somatic motor neurons of the hypoglossal nucleus (12N). All four genes are also expressed in parasympathetic neurons of the facial nerve controlling the lacrimal and salivary glands (Lac/Sal) (third row panels) but had absent or lower expression in the adjacent facial motor nucleus (7N). Allen Brain Atlas images obtained from available postnatal day 56 sections (Celf6, Kcna5, Dgkb) and embryonic day 18.5 sections (Tbx3). Amb sections are sagittal except for Celf6 (coronal). 10N and Lac/Sal sections are sagittal. Bar of 200 μm applies to all sections.

Extended Data Fig. 4 Conservation of ACP and ACV markers across postnatal development.

a, Immunostaining of ACP neurons in the rostral Amb of a neonatal (postnatal day 2) mouse. ACP neurons stained positive for calbindin (red) and stained weakly positive for BChE (cyan). b, Immunostaining of neonatal ACV neurons in the caudal Amb of a postnatal day 2 mouse. ACV neurons stained positive for BChE and negative for calbindin. Bars, 20 μm. c, Map of ACP and ACV neurons in neonatal Amb. Sagittal schematic view showing overlay of soma of all neurons (circles) across all sections spanning a single neonatal (postnatal day 2) Amb that was stained for ACP marker calbindin (dark blue circles) and ACV marker BChE (light blue circles). d, Quantification of absolute numbers of ACP neurons (calbindin+, dark blue) and ACV neurons (BChE+, light blue) per Amb in neonatal mice (mean ± s.d., n = 3 mice). e, Immunostaining of ACP neurons in the rostral Amb of an adult (postnatal day 60) mouse. ACP neurons stained positive for calbindin (red) and stained weakly positive for BChE (cyan). f, Immunostaining of adult ACV neurons in the caudal Amb of a postnatal day 60 mouse. ACV neurons stained positive for BChE and negative for calbindin. Bars, 20 μm. g, Map of ACP and ACV neurons in adult Amb. Sagittal schematic view showing overlay of soma of all neurons (circles) across all sections spanning a single adult (postnatal day 60) Amb that was stained for calbindin (dark blue) and BChE (light blue). Note similarity in marker expression and cell type distribution in neonatal and adult Amb. h, Quantification of absolute numbers of ACP neurons (calbindin+, dark blue) and ACV neurons (BChE+, light blue) per Amb in adult mice (mean ± s.d., n = 3 mice).

Source data

Extended Data Fig. 5 Viral targeting of ACP or ACV neurons.

a, Combined immunostaining and smFISH showing overlap between BChE protein expression (cyan) and Ghsr mRNA expression (purple) in ACV neurons (dashed outlines). Bar, 100 µm. b, Quantification of panel a showing fraction of BChE+ ACV neurons that express Ghsr in Amb (n = 3 mice, 21 neurons total). c, Strategy for targeting ACP or ACV neurons. A Cre-dependent AAV encoding the opsin bReaChES fused to eYFP was delivered to the left or right Amb in Calb1cre mice (to target ACP neurons) or Ghsrcre mice (to target ACV neurons). d, Targeting specificity of AAV-DIO-bReaChES-eYFP vector used for left- and right-sided terminal mapping and optogenetics experiments in Figs. 3–4 (n = 10 Calb1cre, n = 10 Ghsrcre mice, n = 612 neurons total, mean ± s.d.). eYFP expression on the correct side of the brainstem was verified for all mice, and % of population eYFP+ was calculated for the unilateral (injected) Amb. When injected into Calb1cre mice, ACP (calbindin+) neurons were specifically labeled. When injected into Ghsrcre mice, ACV (BChE+) neurons were specifically labeled, though note lower efficiency than the ACP neuron strategy. e, Immunostaining of Rostral Amb ACP neurons in Calb1cre mice injected with AAV-DIO-bReaChES-eYFP vector. Two calbindin+ neurons were eYFP-positive (arrowheads), indicating bReaChES-eYFP expression. f, Immunostaining of caudal Amb ACV neurons in Ghsrcre mice injected with AAV-DIO-bReaChES-eYFP vector. A BChE+ neuron was eYFP-positive (arrowhead), indicating bReaChES-eYFP expression. Bars, 20 μm. g, Distribution of eYFP expression after rostral Amb injection of AAV-DIO-bReaChES-eYFP in a Calb1cre mouse. Note eYFP expression in the Amb external formation (AmbEx) and in overlapping Bötzinger complex (BötC) and pre-Bötzinger complex (preBötC) breathing control regions, with sparing of Amb compact formation (AmbC, esophageal motor neurons), Amb semicompact formation (AmbSc, pharyngeal motor neurons), Amb loose formation (AmbL, laryngeal motor neurons), facial motor nucleus (nVII), retrotrapezoid nucleus (RTN), and lateral reticular nucleus (LRt). Bar, 100 um. h, Sagittal brainstem section showing lack of eYFP expression in dorsal motor nucleus of vagus (10N) and nucleus of the solitary tract (Sol) after AAV-DIO-bReaChES-eYFP injection into Amb in Calb1cre mouse. Bar, 100 um. i, Whole-mount immunostaining showing minimal eYFP expression in the nodose-jugular complex (NJC) after AAV-DIO-bReaChES-eYFP injection into Amb in Calb1cre mouse. Bar, 100 µm. j, Distribution of eYFP expression after caudal Amb injection of AAV-DIO-bReaChES-eYFP in a Ghsrcre mouse. Note eYFP expression in AmbEx, AmbL, and in overlapping BötC and preBötC, similar to Calb1cre mice in panel g but with a more caudal distribution. Bar, 100 um. k, Sagittal brainstem section showing lack of eYFP expression in 10N and Sol after AAV-DIO-bReaChES-eYFP injection into Amb in Ghsrcre mouse. Bar, 100 um. l, Whole-mount immunostaining showing lack of eYFP expression in the NJC after AAV-DIO-bReaChES-eYFP injection into Amb in Ghsrcre mouse. Bar, 100 µm.

Source data

Extended Data Fig. 6 Cardiac GP projection targets of ACP and ACV neurons.

a, Estimated proportions of ganglion neurons within each indicated ganglionated plexus (GP) that receive innervation from a given side and cell type, labeled as in Fig. 3 (n = 2067 neurons total, 2 mice per unilateral cell type). Remaining cells not innervated by ACP or ACV neurons are likely innervated by dorsal motor nucleus of vagus or possibly other ganglion neurons. b, Schematics (based on panel a) of left and right atria (LA and RA) of heart showing innervation of four cardiac GPs (red ovals) by left and right ACP (dark blue) and ACV (light blue) neurons. Thick arrows, dense innervation; thin arrows, sparse innervation. Note left and right ACP neurons innervate same set of GPs, whereas left and right ACV neurons innervated different sets of GPs. Ao, aorta; PA, pulmonary artery; PVs, pulmonary veins; SVC, superior vena cava; IVC, inferior vena cava. c, Immunostaining of the right cardiac GP (dotted outline) after right ACV fibers were labeled with eYFP. The right GP stained positive for vesicular acetylcholine transporter (VAChT), and many eYFP+ ACV fibers innervate ganglion neurons within the GP. d, Immunostaining of the right GP (dotted outline) after right ACP fibers were labeled with eYFP as in Fig. 3. In contrast to right ACV fibers, few eYFP+ fibers from right ACP neurons were found innervating right GP ganglion neurons. Bars, 50 μm.

Source data

Extended Data Fig. 7 Amb optogenetic stimulation in Calb1cre or Ghsrcre mice results in apnea mediated by non-cholinergic neurons.

Data are from same stimulation trials as Fig. 4. a, Schematic of optogenetic activation of left Amb ACP or ACV neurons in anesthetized Calb1cre or Ghsrcre mice. b, Before (-) atropine administration, optogenetic stimulation of left Amb Calb1 neurons (dark blue bars) or left Amb Ghsr neurons (light blue bars) resulted in apnea or reduction in respiratory rate (RR) (n = 5 mice per genotype). After (+) atropine administration, the apnea resulting from left Amb Calb1 or Ghsr neuron stimulation remained fully intact, indicating the effects are mediated by non-cholinergic neurons. c, Schematic of optogenetic activation of right Amb ACP or ACV neurons in anesthetized Calb1cre or Ghsrcre mice. d, Before atropine administration, optogenetic stimulation of right Amb Calb1 neurons (dark blue bars) or right Amb Ghsr neurons (light blue bars) resulted in apnea (n = 5 mice per genotype). After atropine administration, the apnea resulting from right Amb Calb1 or Ghsr neuron stimulation remained fully intact, indicating the effects are mediated by non-cholinergic neurons. These respiratory effects are likely mediated by opsin expression in non-cholinergic interneurons of the pre-Bötzinger complex, an important breathing control region which overlaps significantly with Amb, contains Calb1- and Ghsr-expressing interneurons15,52, and where optogenetic stimulation of interneurons is known to result in apnea53. e, Immunostaining of ACP neurons in Amb in a Calb1cre mouse injected with AAV-DIO-bReaChES-eYFP in Amb where injection failed to target ACP neurons (red), but still targeted nearby interneurons and fibers (green). Note lack of eYFP expression in ACP cell bodies (arrowheads). Bar, 25 μm. f, Respiratory rate (RR) and heart rate (HR) during optogenetic stimulation of interneurons surrounding ACP neurons in Calb1cre mouse from panel e. Note decrease in respiratory rate with optogenetic stimulation of interneurons (yellow bar, 40 Hz), with no changes in heart rate. bpm, breaths per minute (for RR) or beats per minute (for HR). g, Immunostaining of ACV neurons in Amb in Ghsrcre mouse injected with AAV-DIO-bReaChES-eYFP in Amb where injection failed to target ACV neurons (cyan), but still targeted nearby interneurons and fibers (green). Note lack of eYFP expression in ACV cell bodies (arrowheads). Bar, 25 μm. h, Respiratory rate (RR) and heart rate (HR) during optogenetic stimulation (yellow bar, 40 Hz) of interneurons surrounding ACV neurons in Ghsrcre mouse from panel g. Note decrease in respiratory rate with optogenetic stimulation of interneurons (yellow bar), with no changes in heart rate. bpm, breaths per minute (for RR) or beats per minute (for HR).

Source data

Extended Data Fig. 8 c-Fos negative control studies and heart rate response to dive reflex.

a, Immunostaining of ACP neurons in rostral Amb following vehicle injection (see Fig. 5). Note ACP neurons (calbindin+; white arrowheads) are c-Fos negative. b, Immunostaining of ACV neurons in caudal Amb following vehicle injection. Note ACV neurons (BChE+; white arrowheads) are c-Fos negative. c, Immunostaining of ACP neurons in rostral Amb following isoflurane anesthesia without nasal immersion. Note ACP neurons (calbindin+; white arrowheads) are c-Fos negative. d, Immunostaining of ACV neurons in caudal Amb following isoflurane anesthesia without nasal immersion. Note ACV neurons (BChE+, white arrowheads) are c-Fos negative. Bars, 20 μm. e, Example heart rate trace recorded by ECG during dive reflex activation for Fig. 5 experiments. Isoflurane-anesthetized mouse underwent nasal immersion (arrow, start of dive) for 10 s. Bradycardia and AV block were observed during nasal immersion, and heart rate returned to baseline following cessation of immersion.

Extended Data Fig. 9 ACP neurons are not activated early after phenylephrine injection.

a, Experimental time course paradigm. Phenylephrine (PE) (10 mg/kg, IP) was injected into awake mice and mice were sacrificed to perform immunostaining for c-Fos and the indicated AmbCardiac markers at the indicated time points following PE injection. b–e, ACP neurons (calbindin+, arrowheads) did not express c-Fos (red) at any of the time points (30–120 min) following PE injection. Bars, 20 µm.

Extended Data Fig. 10 Clonal analysis of ACP neurons.

a, ACP clonal labeling experiment in Ghsrcre mouse injected with AAV-DIO-eYFP. Left, immunostaining of the soma (dashed outline) in the right Amb of the single eYFP-labeled ACP neuron in this mouse (Clone #2); note it co-stained positive for calbindin (cyan) and negative for BChE (red), confirming ACP identity. Bar, 20 μm. Right, map of ACP neurons (dark blue fill circles) in right Amb (overlay of all sagittal sections of right Amb) showing location of the eYFP-labeled ACP clone (green fill circle). Terminals of the ACP clone (green fibers) were mapped in the heart (left) and lung (right), where it was found to innervate parasympathetic ganglia in both organs (red fill ovals/circles, targeted ganglia). LA, left atrium; RA, right atrium; Ao, aorta; PA, pulmonary artery; PVs, pulmonary veins; SVC, superior vena cava. b, Immunostaining of parasympathetic cardiac ganglion (Ganglion 1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #2. Note innervated neuron stained positive for cholinergic marker VAChT (red) and receives innervation from a cholinergic (red), calbindin-positive (cyan) fiber labeled with eYFP (green). Bar, 10 μm. c, Immunostaining of lung parasympathetic ganglion (Ganglion L1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #2. Note innervated neuron stained positive for neuronal marker Tuj1 (red) and receives innervation from a calbindin-positive (cyan), eYFP-positive (green) fiber. Bar, 10 μm. d, ACP clonal labeling experiment in Calb1cre mouse injected with limiting dose of AAV-FLEX-GFP. Left, immunostaining of the soma (dashed outline) in the L Amb of the single GFP-labeled ACP neuron in this mouse (Clone #3); note it co-stained positive for calbindin (cyan) and negative for BChE (red), confirming ACP identity. Bar, 20 μm. Right, map of ACP neurons (dark blue fill circles) in left Amb showing location of the GFP-labeled ACP clone (green fill circle, Clone #3). Terminals of the ACP clone (green fibers) were mapped in the heart (left) and lung (right), where it was found to innervate parasympathetic ganglia in both organs (red fill ovals/circles, targeted ganglia). e, Immunostaining of parasympathetic cardiac ganglion (Ganglion 1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #3. Note innervated neuron stained positive for cholinergic marker VAChT (red) and receives innervation from a cholinergic (red), calbindin-positive (cyan) fiber labeled with GFP (green). Bar, 10 μm. f, Immunostaining of lung parasympathetic ganglion (Ganglion RMd1) showing a ganglion neuron (dashed outline) innervated by ACP Clone #3. Note innervated neuron stained positive for neuronal marker Tuj1 (red) and receives innervation from a calbindin-positive (cyan), GFP-positive (green) fiber. Bar, 10 μm.

Supplementary information

Supplementary Information

This file contains a full guide to Supplementary Tables 1–7.

Reporting Summary

Supplementary Table 1

Genes enriched in AmbLaryngeal neurons (retrograde labelled from laryngeal muscle) relative to AmbCardiac (retrograde labelled from heart) (P < 0.05, Wilcoxon rank sum test with Bonferroni correction).

Supplementary Table 2

Genes enriched in AmbCardiac neurons (retrograde labelled from laryngeal muscle) relative to AmbLaryngeal (retrograde labelled from heart) (P < 0.05, Wilcoxon rank sum test with Bonferroni correction).

Supplementary Table 3

Selected genes differentially expressed between AmbCardiac and AmbLaryngeal neurons (from Supplementary Tables 1, 2) arranged by function.

Supplementary Table 4

Genes enriched in ACP relative to ACV (P < 0.05, Wilcoxon rank sum test with Bonferroni correction).

Supplementary Table 5

Genes enriched in ACV relative to ACP (P < 0.05, Wilcoxon rank sum test with Bonferroni correction).

Supplementary Table 6

Selected genes differentially expressed between ACP and ACV neurons (from Supplementary Tables 4, 5) arranged by function.

Supplementary Table 7

Projection targets of single ACP neurons.

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Veerakumar, A., Yung, A.R., Liu, Y. et al. Molecularly defined circuits for cardiovascular and cardiopulmonary control. Nature 606, 739–746 (2022). https://doi.org/10.1038/s41586-022-04760-8

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