Objective Integration 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.
Design Unbiased 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.
Results Transcriptomic 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.
Conclusions We 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.
- visceral sensory neurones
- single-cell RNA sequencing
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Contributors JRFH, DCB, WJW and EStJS designed the study. JRFH, TST, GC and ALW undertook experiments. JRFH, TST, AG, KB, DCB and GM analysed the data. JRFH, DCB and EStJS wrote the manuscript in conjunction with all other authors.
Funding This work was supported by Neusentis (Pfizer) in the form of salaries for authors (JRFH, ALW, AG, WJW and GM), but did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Additionally, this work is supported by a Rosetrees Postdoctoral Grant (A1296) awarded to JRFH and EStJS, Medical Research Council Grant (G0900907) to DCB, University of Cambridge Vice Chancellor’s Award (TST), Arthritis Research UK Grant to GC and EStJS (RG 20930). KB is funded by a Cambridge Cancer Centre studentship.
Provenance and peer review Not commissioned; externally peer reviewed.
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