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A Single Neuron Scale Molecular Phenotype Map of the Right Atrial Ganglionic Plexus in the Pig Heart

ABSTRACT: We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion - the right atrial ganglionic plexus (RAGP) that is a critical mediator of vagal control of the sinoatrial node (SAN) activity. We developed a 3D representation of RAGP with extensive mapping of neurons and used neuronal tracing to identify the spatial distribution of the subset of neurons that project to the SAN. RNAseq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to CNS neuronal subtypes. High-throughput qPCR of hundreds of laser capture microdissected single neurons led to a surprising finding that cholinergic and catecholaminergic neuronal markers Th and Chat were correlated, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. Interestingly, no single gene or module was an exclusive marker of RAGP neuronal connectivity to SAN. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP, informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease.

ORGANISM(S): Sus scrofa

PROVIDER: GSE154119 | GEO | 2021/05/28

REPOSITORIES: GEO

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Project description:As part of the NIH SPARC program efforts to study the intrinsic cardiac nervous system, we developed the first comprehensive molecular phenotyping data on the right atrial ganglionated plexus (RAGP) within the intrinsic cardiac nervous system of the pig. We collected hundreds of single neurons from the RAGP and assayed these for expression of a wide range of genes relevant to neuronal functions. The sinoatrial node (SAN) of 4 pigs were injected with FastBlue, a retrograde tracer, labeling neurons projecting to the SAN. The RAGP of those pigs were then collected and sectioned. Both SAN-projecting and non-SAN-projecting single neurons in RAGP were collected through laser capture microdissection (LCM), allowing for spatial mapping of all collected samples. Over 400 single neurons were collected (n=4 animals) and each neuron was assayed for over 300 genes using high throughput microfluidic qPCR using BioMark. Neuronal phenotypes were distinguished by multivariate analysis showing patterns of network activity between multiple genes. Interestingly, these phenotypes show no spatial preferences within the RAGP. Our results revealed extensive combinatorial expression of neurotransmitters across the RAGP neuronal phenotypes. Additionally, there was a large overlap in expression profiles of SAN-projecting and non-SAN-projecting neurons, without any single gene or module acting as a distinguishing marker between the connectionally labeled groups. Previous studies have focused on cholinergic and catecholaminergic processes, showing evidence for extensive protein expression of cholinergic markers in RAGP. Our findings are in stark contrast to these results and demonstrate high gene expression correlation between cholinergic and catecholaminergic markers such as tyrosine hydroxylase (Th) and choline acetyltransferase (ChAT) in single neurons. This finding suggests that cells are poised for a rapid shift in neurotransmitter signaling as well as the possibility for post transcriptional regulation of neuron phenotype plasticity. Our present findings significantly expand the list of neuromodulatory peptides and their receptors known to be expressed in RAGP. For example, neuropeptide Y and somatostatin showed distinct co-expression patterns with their respective receptors with significant non-overlapping expression of transmitters and cognate receptors across single neurons. Taken together, our results reveal a complex organization of neuronal networks within RAGP-SAN complex that is not entirely governed by connectivity or spatial location, with combinatorial patterns of local paracrine networks with potential influence on cardiac function.

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A Single Neuron Scale Molecular Phenotype Map of the Right Atrial Ganglionic Plexus in the Pig Heart

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