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JNEUROSCI.1426-20.2020.Full.Pdf Research Articles: Cellular/Molecular Unique molecular characteristics of visceral afferents arising from different levels of the neuraxis: location of afferent somata predicts function and stimulus detection modalities https://doi.org/10.1523/JNEUROSCI.1426-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1426-20.2020 Received: 2 June 2020 Revised: 30 July 2020 Accepted: 7 August 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Unique molecular characteristics of visceral afferents arising from different levels of the 2 neuraxis: location of afferent somata predicts function and stimulus detection modalities 3 Running Title: Unique molecular profile of visceral afferents 4 Kimberly A. Meerschaert1,2, Peter C. Adelman 3, Robert L. Friedman 1,2, Kathryn M. Albers 1,2, H. 5 R. Koerber 1,2 and Brian M. Davis 1,2,# 6 1 Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, 7 Pennsylvania; 2 Pittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, 8 Pennsylvania; 3 Afiniti, Washington D.C. 9 #For correspondence: Brian M. Davis, University of Pittsburgh, Department of Neurobiology, 10 200 Lothrop Street, Pittsburgh, PA 15216, [email protected], Ph: 412-648-9745 11 Number of pages: 36 12 Number of figures and tables: 7 Figs., 3 Tables 13 Number of words for Abstract - 250, Significance Statement- 101; Introduction – 647, and 14 Discussion - 1489 15 All authors have no conflict of interest. 16 Acknowledgements: The authors acknowledge and thank Mr. Christopher Sullivan for expert 17 technical support and mouse husbandry. 18 Author contributions: KAM, conception and design, acquisition of data, analysis and 19 interpretation of data, drafting and revising the article. PCA and RLF, acquisition of data, 20 analysis and interpretation of data. KMA, HRK and BMD, conception and design, analysis and 21 interpretation of data, drafting and revising the article. 1 22 Supported by NINDS NS023725, NS096705 (HRK), NIAMS AR069951 (KMA), NIDDK 23 DK124955 (BMD), OT2-OD023859 (Marthe J. Howard PD, BMD PI) 24 25 Keywords: Dorsal root ganglia, nodose ganglia, primary afferent, colon, bladder 26 Abbreviations: DRG, dorsal root ganglia; NG, nodose ganglia; RT-qPCR, real-time quantitative 27 polymerase chain reaction; TL, thoracolumbar; LS, lumbosacral 28 2 29 Abstract 30 Viscera receive innervation from sensory ganglia located adjacent to multiple levels of the 31 brainstem and spinal cord. Here we examined whether molecular profiling could be used to 32 identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and 33 nodose ganglia (NG) in male and female mice. Profiling of TL and LS bladder afferents was 34 also done. Visceral afferents were back-labeled using retrograde tracers injected into proximal 35 and distal regions of colon or bladder, followed by single cell RT-qPCR and analysis via an 36 automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for 37 colon) based on their established role in stimulus detection, regulation of sensitivity/function or 38 neuroimmune interaction. A total of 132 colon afferents (from NG, TL, and LS) and 128 bladder 39 afferents (from TL and LS) were analyzed. Retrograde labeling from the colon showed NG and 40 TL afferents innervate proximal and distal regions of the colon whereas 98% of LS afferents 41 only project to distal regions. There were clusters of colon and bladder afferents, defined by 42 mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was 43 found. In addition, transcriptionally, NG colon afferents were almost completely segregated 44 from colon TL and LS neurons. Furthermore, colon and bladder afferents expressed genes at 45 similar levels, although different gene combinations defined the clusters. These results indicate 46 that genes implicated in both homeostatic regulation and conscious sensations are found at all 47 anatomic levels, suggesting afferents from different portions of the neuraxis have overlapping 48 functions. 49 50 Significance Statement: Visceral organs are innervated by sensory neurons whose cell 51 bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the 52 colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, 53 where sensation and pain is mediated by spinal afferents and fear and anxiety (the affective 3 54 aspects of visceral pain) are the domain of nodose afferents. Transcriptomic analysis 55 performed here reveals that genes implicated in both homeostatic regulation and pain are found 56 in afferents across all ganglia types, suggesting that conscious sensation and homeostatic 57 regulation is the result of convergence, and not segregation, of sensory input. 58 Introduction 59 Visceral organs receive sensory innervation from primary sensory neurons arising from multiple 60 levels of the neuraxis. Depending on the organ, innervation can originate from vagal afferents 61 (including afferents in the jugular and nodose (NG) ganglia), thoracolumbar (TL) spinal afferents 62 (arising from lower thoracic and upper lumbar dorsal root ganglia (DRG)) or lumbosacral (LS) 63 spinal afferents (arising from lower lumbar and upper sacral DRG). Our hypotheses to account 64 for why viscera requires monitoring by multiple populations of afferents include: a) that different 65 levels of innervation are involved in different qualitative aspects of stimulus detection, including 66 pain, b) that because the first synapse is on CNS neurons associated with sympathetic or 67 parasympathetic circuits, different afferent populations provide the basis for integration of 68 autonomic/homeostatic functions, or c) that the different levels play complementary roles in 69 immune modulation. 70 With regard to shaping the nature of the visceral sensory experience, it had been hypothesized 71 that spinal afferents, but not vagal afferents, relay nociceptive sensations (e.g., stabbing, 72 burning, cramping), whereas vagal afferents underlie affective aspects of visceral pain including 73 depression, anxiety, nausea and fear (Altschuler et al., 1993; Berthoud and Neuhuber, 2000; 74 Grundy, 2002; Sengupta, 2009). However, recent evidence has shown that vagal afferents may 75 have a significant role in nociception and pain (Lee et al., 2003; Kang et al., 2004; Yu et al., 76 2005; Bielefeldt et al., 2006; Canning et al., 2006; Nassenstein et al., 2008; Taylor-Clark et al., 77 2009). Even spinal afferents from different levels may have specific roles; colonic afferents 78 arising from the TL level have been reported to be important for inflammatory pain but not acute 4 79 pain, whereas LS colonic afferents are proposed to be involved in both acute and inflammatory 80 pain (Traub et al., 1994; Traub, 2000; Wang et al., 2005). 81 In terms of homeostasis, spinal visceral afferents that innervate colon and bladder have cell 82 bodies in both TL and LS DRG, projecting to organs they innervate along sympathetic (TL) or 83 parasympathetic (LS) splanchnic nerves and terminate broadly in the spinal cord, including in 84 the region of sympathetic (TL) or parasympathetic (LS) preganglionic neurons (Harrington et al., 85 2019; Smith-Edwards et al., 2019). In contrast, vagal afferents that innervate the colon (and 86 possibly bladder (Herrity et al., 2014)) have cell bodies in the nodose ganglia, run in the vagus 87 nerve with preganglionic parasympathetic fibers and terminate in the brainstem nucleus tractus 88 solitarius (NTS) that in turn projects to nuclei in the brainstem, thalamus and cortex (Norgren, 89 1978; van der Kooy et al., 1984). Thus, redundant sensory input to the CNS could be informing 90 different components of the autonomic nervous system. 91 Afferent/immune interactions are also critical for gut function and homeostasis, i.e., the vagus 92 nerve has a crucial role in the anti-inflammatory reflex (Tracey, 2002), with vagal afferent 93 response to cytokine release as the first step in this reflex. Primary afferents (vagal and spinal) 94 release multiple peptides including calcitonin gene-related peptide (CGRP) and substance P 95 that bind receptors on immune cells, allowing direct afferent and immune communication (Brain 96 and Williams, 1985; Caceres et al., 2009; Altmayr et al., 2010; Riol-Blanco et al., 2014; Cohen 97 et al., 2019). Ablation or silencing of primary afferents (vagal and spinal) also can modulate the 98 immune response across multiple organs, including the lungs, small intestine and colon (Engel 99 et al., 2011; Baral et al., 2018; Lai et al., 2020). 100 In this study we examined how genes required for sensation, homeostasis and neuroimmune 101 regulation are divided among different visceral afferents. Single cell RT-qPCR analysis 102 combined with automated hierarchical clustering (AHC) of afferents innervating the bladder and 103 colon were carried out. Results show that location matters; that genes expressed by an afferent 5 104 and where that afferent terminates (especially for the colon) is determined by the ganglionic 105 location. Furthermore, the clustering of unique genes to afferents from
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