Single-cell RNAseq reveals seven classes of colonic sensory neuron

Abstract

ObjectiveIntegration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons.DesignUnbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca2+-imaging.ResultsTranscriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca2+-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation.ConclusionsWe identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development.

Endogenous control of inflammatory visceral pain by T cell‐derived opioids in IL‐10‐deficient mice

“…The muscle layer and mucosa both innervated by spinal afferents, that can be inhibited by opioids, 28,[39][40][41] are infiltrated by lymphocytes. The muscle layer and mucosa both innervated by spinal afferents, that can be inhibited by opioids, 28,[39][40][41] are infiltrated by lymphocytes.…”

“…A number of colon biopsies from IL-10 −/− colitis mice exhibit transmural damage including an inflammation of the muscularis. The muscle layer and mucosa both innervated by spinal afferents, that can be inhibited by opioids, 28,[39][40][41] are infiltrated by lymphocytes. Based on previous studies reporting that T lymphocytes may specifically migrate toward afferent nerve endings innervating the inflammatory site including mucosa and muscularis, it could be speculated that T cell-mediated pain relief is still observed because of an optimal topographic distribution of opioid-producing CD4 + T lymphocytes at the vicinity of sensory nerves.…”

A pharmacological interactome platform for discovery of pain mechanisms and targets

“…RNA sequencing (RNA-seq) experiments have defined tissue-wide and cell-specific transcriptomes for much of the body in both mice (Usoskin et al, 2015;Schaum et al, 2018;Zeisel et al, 2018) and humans (Mele et al, 2015;Ray et al, 2018). Cell profiling experiments on normal and diseased tissues have identified key molecular players in an increasing number of disease processes (Roy et al, 2018), including disorders with a strong pain component (Hockley et al, 2018;Kuo et al, 2019). However, these studies mostly focus on gene expression within a specific tissue or across cell types in a tissue and do not characterize how multiple tissues may interact to promote disease.…”

“…Then, we performed 3 case studies to demonstrate the utility of this tool for identifying potential drivers of pain states. First, we used single cell RNA-seq (scRNA-seq) data from colon-innervating nociceptors in the mouse (Hockley et al, 2018) to illustrate how these neurons interact with normal and inflamed enteric glial cells (Delvalle et al, 2018). Second, we examined how human DRG (hDRG) neurons (Ray et al, 2018;North et al, 2019) interact with macrophages taken from the joints of people with rheumatoid arthritis (RA) (Kuo et al, 2019).…”

Intra-articular AAV-PHP.S mediated chemogenetic targeting of knee-innervating dorsal root ganglion neurons alleviates inflammatory pain in mice

“…Peripheral sensitization, manifested by an increase in excitability of dorsal root ganglion (DRG) neurons, underlies many chronic pain pathologies, such as inflammatory arthritis (1). There is great heterogeneity of DRG neurons based upon gene expression (2,3) and functional attributes (4), and this heterogeneity is further compounded by target innervation (5,6). This variation in DRG neurons offers a unique opportunity to selectively tune the excitability of a distinct subset of DRG neuron in order to provide pain relief with reduced side-effects.…”