Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies

Pradeep S Rajendran,Heike Münzberg,Guy Salama,Rosemary C Challis,John D Tompkins,Benjamin E Deverman,Jeffrey L Ardell,Ken Y Chan,Sarah Hiyari,Peter Hanna,Charless C Fowlkes,Beth A Gabris-Weber,Viviana Gradinaru,Maria C Jordan,Alon Greenbaum,Kalyanam Shivkumar

doi:10.1038/s41467-019-09770-1

Abstract

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

Highlights

Heart rate is under the precise control of the autonomic nervous system
In contrast to whole-mount stained hearts, innervation was seen throughout the entire thickness of the myocardium in immunolabelingenabled three-Dimensional Imaging of Solvent-Cleared Organs (iDISCO)-cleared hearts, with large-diameter nerve fiber bundles located near the epicardium and smaller fiber bundles in the mid-myocardium and endocardium (Fig. 1c, Supplementary Fig. 1, and Supplementary Movie 3)
These bundles coursed perpendicular to the atrioventricular (AV) groove and branched into smaller fiber bundles as they progressed towards the apex

Summary

Introduction

Heart rate is under the precise control of the autonomic nervous system. the wiring of peripheral neural circuits that regulate heart rate is poorly understood. We develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. Anatomical and functional maps of these fundamental cardiac circuits are needed to understand physiology, characterize remodeling in disease (e.g., sick sinus syndrome14), and develop novel therapeutics These efforts have been hindered by a shortage of tools that target the peripheral nervous system (PNS) with specificity and precision. We develop a clearing-imaging-analysis pipeline to visualize innervation of whole hearts in 3D and employ a multitechnique approach, which includes AAV-based sparse labeling and tracing, retrograde neuronal tracing with cholera toxin subunit B (CTB), and optogenetics with simultaneous physiological measurements, to map peripheral parasympathetic and sympathetic neural circuits that regulate heart rate in mice

Methods

Results

Conclusion