Single Cell Gene Expression Analysis and 3‐D Mapping of Cardiac Ganglia

The intrinsic nervous system within the heart influences cardiac function at both physiological and molecular levels. The human cardiac ganglionic innervation organized into functionally distinct ganglia is composed of 14,000 neurons. Recent studies suggest that ganglia work together in an interaction network to control cardiac function. It has been shown that cardiac neurons are functionally heterogeneous and that the specific neuronal network that influences each neuron can also be broadly heterogeneous. Functional understanding of the complexity within cardiac intrinsic nervous system and its communication to and from the autonomic centers of the central nervous system is incomplete. Additionally, there is sparse knowledge of the spatial organization of these neuronal phenotypes within and among cardiac ganglia. In this study we aim to identify distinct spatially organized neuronal transcriptional phenotypes with high resolution using a novel combination of immunohistochemistry ‐ labeling and 3‐D reconstruction of the whole rat heart along with transcriptomic data of single neuron samples collected from rat hearts. Block face imaging of several rat hearts along with cresyl‐violet stained sections is being used to construct a 3‐D atlas of the rat heart with ontological information and spatial neuronal locational information (in collaboration with MBF Biosciences). We are collecting single neurons as well as neuron pools (comprising of 5 or 10 cells) using Laser Capture Microdissection from rat hearts. These samples are being processed for transcriptome analysis (multiplexed RNA‐seq). We are using BioMark multiplex RT‐qPCR system to validate our transcriptional data obtained from RNA Seq. Transcriptional data along with spatial location of hundreds neurons per heart thus obtained will enable us to map molecular profiles of cardiac ganglia heterogeneity. We have demonstrated the ability to identify molecular phenotypes of neurons through preliminary examination of ~250 laser captured neurons using multiplex RT‐qPCR. We have also generated a more targeted list of cardiac ganglia specific genes through microarray experiments comparing neuron‐enriched sections of the heart with unenriched myocardium. Multiplexed RT‐qPCR data shows distinct molecular phenotypes emerge even with consideration of only ~40 genes. We were able to identify dopaminergic, catecholaminergic, and two types of cholinergic neurons from these data. Within each larger phenotype there exists heterogeneity of transcriptional states that support the need for further investigation. Along with circuit relevance of ganglia projections we will be able to generate a cardiac neuro‐reference framework atlas with neuronal distribution, density of innervation, receptive (sensory) or motor fields and terminal structures of axons from different peripheral ganglia and vagal nuclei. Our study will provide a new holistic view of vagal and spinal afferent, autonomic efferent (sympathetic and parasympathetic), and cardiac ganglionic innervation of the heart in 3‐D whole heart qualitatively and quantitatively. Support or Funding Information SPARC‐OT2‐OD023848 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .