Abstract
In the past few years, there has been extraordinary interest in how the gut communicates with the brain. This is because substantial and gathering data has emerged to suggest that sensory nerve pathways between the gut and brain may contribute much more widely in heath and disease, than was originally presumed. In the skin, the different types of sensory nerve endings have been thoroughly characterized, including the morphology of different nerve endings and the sensory modalities they encode. This knowledge is lacking for most types of visceral afferents, particularly spinal afferents that innervate abdominal organs, like the gut. In fact, only recently have the nerve endings of spinal afferents in any visceral organ been identified. What is clear is that spinal afferents play the major role in pain perception from the gut to the brain. Perhaps surprisingly, the majority of spinal afferent nerve endings in the gut express the ion channel TRPV1, which is often considered to be a marker of “nociceptive” neurons. And, a majority of gut-projecting spinal afferent neurons expressing TRPV1 are activated at low thresholds, in the “normal” physiological range, well below the normal threshold for detection of painful sensations. This introduces a major conundrum regarding visceral nociception. How should we define a “nociceptor” in the gut? We discuss the notion that nociception from the gut wall maybe a process encrypted into multiple different morphological types of spinal afferent nerve ending, rather than a single class of sensory ending, like free-endings, suggested to underlie nociception in skin.
Introduction
Nociceptors are primary afferent neurons that are capable of detecting and responding to potentially damaging stimuli (). In the skin, different types of sensory nerve endings have been well characterized () and cutaneous nociceptors are defined as a unique class of afferent fiber that is morphologically considered a “free-ending” (). Unlike the skin, however, where the functional role of the different morphological types of sensory nerve endings are well characterized, our understanding of the functional role of the different morphological types of extrinsic sensory nerve endings in visceral organs, like the gut is limited. Even less clear is which sensory nerve endings in the gut encode nociceptive stimuli.
A variety of stimuli can activate sensory nerve endings from within the gut wall which initiate the sensation of pain. The visceromotor response (VMR) to colorectal distension (; ; ) or bladder distension () is commonly used as a model for visceral pain measurements. The VMR is a complex neural reflex response from the visceral organ to the central nervous system (CNS). The motor response is contraction of the abdominal muscles (). Mutant mice lacking the TRPV1 channel have reduced VMR distension-evoked pain sensations following colorectal distension (). Hence, there is sound evidence to suspect that Trvp1 expressing spinal afferents encode nociceptive stimuli from the gut to the CNS.
Nociception in the gut
There is no doubt that in vertebrate animals, noxious or potentially damaging stimuli applied to the gut can trigger the sensation of pain (; ). However, unlike skin, pain sensations arising from the gut (visceral pain) are poorly localized and poorly defined. There is also an inability in most parts of the gut, particularly the intestine and colon, to discriminate between different types of pain, e.g., temperature (cold versus heat pain) from mechanical distension-induced pain. Regardless of the type of gut pain, what remains mysterious is what type(s) of sensory neuron(s) may encode pain sensations. That is, which neurons are nociceptive and which ones encode non-nociceptive stimuli? There is some evidence that the vagus nerve can encode some noxious stimuli from the upper gut (). However, of the two major sensory nerves that innervate the gut wall (vagal afferents or spinal afferents), there is an overwhelming body of evidence to suggest that sensory neurons arising from spinal dorsal root ganglia (DRG) are the major sensory nerves encoding noxious stimuli from the gut to the brain (; ). Recent studies have shown that by surgically removing specific populations of DRG, pain reflex responses to mechanical distension of the colon can be ablated (). This occurs whilst leaving all other sensory and motor nerves intact ().
Which spinal afferents contribute to the sensation of pain?
Extracellular recordings have been made from spinal afferents that lie between the colon and spinal cord (, ; , ; ; , ) and intracellular recordings from spinal afferent neurons in DRG that directly communicate with intact colon in ex vivo preparations (; ). When extracellular recordings are made from colonic afferents five distinct classes of afferent fiber can be distinguished, based on mechanical stimuli using von Frey hair probing and/or stretch (). Intracellular recordings from the nerve cell bodies of colon-projecting spinal afferents has revealed primarily high and low threshold firing afferents (; ) many of which are peptidergic and express the alpha isoform of the calcitonin gene related peptide (CGRPα) (). Interestingly, when electrophysiological recordings have been made from colon-projecting spinal afferents, the vast majority have been shown to generate action potentials following distension at low thresholds, referred to as low responders (; ). And, these axons are exclusively C-fibers (), with low conduction velocities. Another study found similar findings (), in that most colon-projecting DRG neurons are activated at low mechanical distension thresholds and over a wide-dynamic range, that includes the both innocuous (non-painful) stimuli and noxious levels of distension pressure ().
Retrograde tracing from DRG revealed that >80% of thoracolumbar DRG neurons express TRPV1 (; ) and 50% of retrogradely labeled lumbosacral DRG neurons that project to colon express TRPV1 (). TRPV1 expression occurred in primarily low frequency responding neurons (87% were TRPV1 +) while only 13% of high frequency responding neurons were TRPV1 + (). When the nerve endings of spinal afferents have been identified in the colon, that vast majority are peptidergic (CGRPα +) and express TRPV1 ().
In the gut, only recently have we been able to visualize with clarity the morphological features of the different types of the nerve endings of spinal afferents by anterograde tracing from DRG (, ,). Using this method, spinal afferent endings have been identified in myenteric ganglia as intraganglionic varicose endings (). On rare occasions intraganglionic lamellar endings (IGLEs) can arise from spinal afferents with cell bodies in DRG (). In the skin, “free nerve endings” are unspecialized bare nerve endings that lack encapsulation at their terminals and lack complex morphologies (). Whilst spinal afferent nerve endings in the gut are also unencapsulated (similar to free-endings in skin), they rarely exhibit simple morphologies (). In fact, spinal afferent endings in the gut exhibit an extraordinarily complex array of different morphologies. Recently, we identified that a single DRG neuron can give rise to different morphological types of nerve ending in different anatomical layers of the gut wall (,). Hence, assigning the nomenclature of “free-ending” for all visceral afferents is a broad misnomer.
Can we distinguish intrinsic sensory nerve endings from extrinsic sensory nerve endings?
A major complication with understanding the extrinsic sensory innervation of the gut, is that the gut itself is unique and has evolved not only with its own nervous system, (known as the enteric nervous system, ENS) (; ), but also its own intrinsic sensory neurons (; ), that express the beta isoform of the calcitonin gene relate peptide (CGRPβ) (; ; ). However, it is currently not possible to use CGRP antisera to differentiate the nerve endings of intrinsic sensory neurons from extrinsic sensory nerves. It is also unclear whether intrinsic sensory neurons provide any collateral projections onto the nerve endings of spinal afferents that also innervate myenteric ganglia. Interestingly, it has been proposed that intrinsic sensory neurons in the myenteric plexus may interact with vagal afferent terminals in the gut-brain axis ().
Can we identify nociceptors in the gut using antibodies?
The problem with using terminology like “visceral nociceptors” in the gut is that well accepted nociceptive ion channels, like TRPV1 are highly expressed in the vast majority (∼90%) of spinal afferent neuron that innervate the colon (). In this regard, in both the colon and bladder, the definition of a visceral afferent as a “nociceptive neuron” based solely on expression of TRPV1 is unhelpful. From our experience, there is no neurochemical marker to date, that can discriminate between different populations of spinal afferent nerve endings in the gut, let alone nerve endings that maybe nociceptors. While it is well accepted that nociception from the gut involves TRPV1 + primary afferent neurons (), it is unclear whether a single neurochemical class of TRPV1 + nociceptor or single morphological class of spinal afferent ending exclusively encodes nociception from the gut. This seems highly unlikely, given the fact that most spinal afferents to the colon are peptidergic () and express Trvp1 () and that low levels of stretch to the colon activate spinal afferents that indeed also respond to capsaicin ().
Can we identify nociceptors in the gut by using morphological studies of transgenic reporter mice?
Transgenic reporter mice have been useful for bulk labeling of large populations of axons in peripheral organs, like the gut. However, they have not been useful for identifying the detailed morphology of the nerve endings of putative “nociceptors” in any visceral organs. This is because firstly, many of the neurochemical markers expressed in spinal afferents are also expressed in vagal afferents or enteric neurons, making interpretation of the origin of the cell bodies of the labeled axons uncertain. And, as mentioned, in reporter mice, labeling of so many axons precludes identification of the fine morphological features that may arise from a single primary afferent. The use of intersectional neurogenetics to narrow the targeted populations in transgenic mice by excluding enteric neuronal populations may overcome this issue in the coming years as the approach becomes more popular. However, all reporter mice will likely label numerous axons that overlap each other making it very difficult to resolve endings that arise from one primary afferent neuron.
Identifying spinal afferent nerve endings using anterograde tracing from DRG
Anterograde technique from single DRG in live mice has enabled unequivocal identification of spinal afferent nerve endings (). Using this approach, the spinal afferent endings have now been identified in bladder (), colon (), stomach (), bone (), and uterus (). What became immediately clear from these studies in gut was that there was a far greater complexity and diversity of different types of endings than we expected. It was also clear that a single DRG neuron did not give rise to sensory endings in only one layer of the gut. In fact, tracing from single DRG neurons revealed that single afferents that projected to the colon had nerve terminal endings in the circular muscle and myenteric ganglia () and another population gave off nerve endings in the myenteric ganglia, submucosa and mucosa (). Other classes have yet to be identified using this technique.
Surgically removing DRG from animals to identify pain pathways
In a recent study, a survival surgical technique was developed to remove specific populations of DRG unilaterally or bilaterally, from live mice (). This approach had the major advantage of not interfering with any other afferent of efferent nerve pathways, only spinal afferents, in live mice. When the lumbosacral DRGs (L5-S2) were surgically removed from mice using this approach, the distension-evoked pain pathway (VMR) from the rectum was abolished (). This revealed important information that although thoracolumbar DRG innervate (to a small degree) the rectum, the functional pain reflex response (VMR) to rectal distension is abolished if the lumbosacral DRG are removed, but thoracolumbar DRG are retained (). This is significant because the lumbosacral DRG pathway to the rectum in mice is primarily a low threshold, wide dynamic range sensory pathway.
Taken together, it is suggested that there may well be no exclusive nociception-dedicated primary afferent neuronor transduction site(s) in the viscera. Rather, nociception may arise from bulk signaling across all visceral afferent morphological and neurochemical subclasses. Indeed, stimuli such as noxious distension that may recruit afferents with high-distension thresholds inevitably recruit all low-threshold afferents too, which can continue to encode higher firing intensities into the noxious range.
Conclusion
As more scientists are turning their attention to understanding gut-brain communication via primary sensory nerves, it is important we develop a clearer understanding of which class(es) of afferent fibers that lie between the gut and brain may contribute to nociception. This is an essential step for development of novel therapeutics to potentially target visceral (gut) nociception. The discovery large populations of TRPV1 expressing spinal afferent neurons underlie a major component of the mechanically activated pain pathway from colon to brain suggests that low threshold mechanically activated ion channels play major roles in encoding both physiological (non-noxious) and noxious pain signals to the brain. What is clear is that there is an extraordinarily complex array of different morphological types of spinal afferent ending in the gut; and it is becoming increasingly unlikely that a single population of sensory neuron underlies nociception and pain-signaling along the gut-brain axis.
Statements
Data availability statement
The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
Author contributions
NS wrote the manuscript in collaboration with TH, ZX, and HH. All authors corrected and approved the final manuscript.
Funding
This work was supported by a National Health and Medical Research Council of Australia (NHMRC) 1156416 and grants from National Institutes of Health grant R01AA027065 (NINDS, HH), National Institutes of Health grant R01AR077183 (NIAMS, HH), and National Institutes of Health grant R01DK103901 (NIDDK, HH).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
1
BasbaumA. I. B. C. (2009). Science of pain (Vol. 37).Amsterdam: Elsevier.
2
BrierleyS. M.CarterR.JonesW.IIIXuL.RobinsonD. R.HicksG. A.et al (2005a). Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice.J. Physiol.567(Pt 1)267–281. 10.1113/jphysiol.2005.089714
3
BrierleyS. M.HibberdT. J.SpencerN. J. (2018). Spinal afferent innervation of the colon and rectum.Front. Cell Neurosci.12:467. 10.3389/fncel.2018.00467
4
BrierleyS. M.JonesR. C.IIIGebhartG. F.BlackshawL. A. (2004). Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice.Gastroenterology127166–178.
5
BrierleyS. M.JonesR. C.IIIXuL.GebhartG. F.BlackshawL. A. (2005b). Activation of splanchnic and pelvic colonic afferents by bradykinin in mice.Neurogastroenterol. Motil.17854–862. 10.1111/j.1365-2982.2005.00710.x
6
CastroJ.HarringtonA. M.HughesP. A.MartinC. M.GeP.SheaC. M.et al (2013). Linaclotide inhibits colonic nociceptors and relieves abdominal pain via guanylate cyclase-C and extracellular cyclic guanosine 3’,5’-monophosphate.Gastroenterology1451334–46.e1-11. 10.1053/j.gastro.2013.08.017
7
DoddsK. N.KylohM. A.TravisL.BeckettE. A. H.SpencerN. J. (2021). Morphological identification of thoracolumbar spinal afferent nerve endings in mouse uterus.J. Comp. Neurol.5292029–2041. 10.1002/cne.25070
8
FengB.GebhartG. F. (2011). Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum.Am. J. Physiol. Gastrointest. Liver Physiol.300G170–G180. 10.1152/ajpgi.00406.2010
9
FengB.BrumovskyP. R.GebhartG. F. (2010). Differential roles of stretch-sensitive pelvic nerve afferents innervating mouse distal colon and rectum.Am. J. Physiol. Gastrointest. Liver Physiol.298G402–G409. 10.1152/ajpgi.00487.2009
10
FengB.LaJ. H.TanakaT.SchwartzE. S.McMurrayT. P.GebhartG. F. (2012). Altered colorectal afferent function associated with TNBS-induced visceral hypersensitivity in mice.Am. J. Physiol. Gastrointest. Liver Physiol.303G817–G824. 10.1152/ajpgi.00257.2012
11
FuentesI. M.WalkerN. K.PierceA. N.HoltB. R.Di SilvestroE. R.ChristiansonJ. A. (2016). Neonatal maternal separation increases susceptibility to experimental colitis and acute stress exposure in male mice.IBRO Rep.110–18. 10.1016/j.ibror.2016.07.001
12
FurnessJ. B. (2006). The enteric nervous system.Oxford: Blackwell Publishing.
13
FurnessJ. B.RobbinsH. L.XiaoJ.StebbingM. J.NurgaliK. (2004). Projections and chemistry of Dogiel type II neurons in the mouse colon.Cell Tissue Res.3171–12. 10.1007/s00441-004-0895-5
14
GrundyL.EricksonA.BrierleyS. M. (2019). Visceral pain.Annu. Rev. Physiol.81261–284. 10.1146/annurev-physiol-020518-114525
15
HandlerA.GintyD. D. (2021). The mechanosensory neurons of touch and their mechanisms of activation.Nat. Rev. Neurosci.22521–537. 10.1038/s41583-021-00489-x
16
HibberdT. J.KestellG. R.KylohM. A.BrookesS. J.WattchowD. A.SpencerN. J. (2016). Identification of different functional types of spinal afferent neurons innervating the mouse large intestine using a novel CGRPalpha transgenic reporter mouse.Am. J. Physiol. Gastrointest. Liver Physiol.310G561–G573. 10.1152/ajpgi.00462.2015
17
HibberdT. J.YewW. P.DoddsK. N.XieZ.TravisL.BrookesS. J.et al (2022). Quantification of CGRP-immunoreactive myenteric neurons in mouse colon.J. Comp. Neurol.5303209–3225. 10.1002/cne.25403
18
JonesR. C.IIIXuL.GebhartG. F. (2005). The mechanosensitivity of mouse colon afferent fibers and their sensitization by inflammatory mediators require transient receptor potential vanilloid 1 and acid-sensing ion channel 3.J. Neurosci.2510981–10989. 10.1523/JNEUROSCI.0703-05.2005
19
KandelE. R.ShwartzJ. H.JessellT. M. (2000). Principles of neural Science, 4th Edn. New York, NY: McGraw-Hill, 431–450.
20
KunzeW. A.BornsteinJ. C.FurnessJ. B. (1995). Identification of sensory nerve cells in a peripheral organ (the intestine) of a mammal.Neuroscience661–4.
21
KylohM. A.HibberdT. J.CastroJ.HarringtonA. M.TravisL.DoddsK. N.et al (2022). Disengaging spinal afferent nerve communication with the brain in live mice.Commun. Biol.5:915. 10.1038/s42003-022-03876-x
22
LairdJ. M.Martinez-CaroL.Garcia-NicasE.CerveroF. (2001). A new model of visceral pain and referred hyperalgesia in the mouse.Pain92335–342.
23
MalinS. A.ChristiansonJ. A.BielefeldtK.DavisB. M. (2009). TPRV1 expression defines functionally distinct pelvic colon afferents.J. Neurosci.29743–752. 10.1523/JNEUROSCI.3791-08.2009
24
MulderryP. K.GhateiM. A.SpokesR. A.JonesP. M.PiersonA. M.HamidQ. A.et al (1988). Differential expression of alpha-CGRP and beta-CGRP by primary sensory neurons and enteric autonomic neurons of the rat.Neuroscience25195–205. 10.1016/0306-4522(88)90018-8
25
Perez-BurgosA.MaoY. K.BienenstockJ.KunzeW. A. (2014). The gut-brain axis rewired: Adding a functional vagal nicotinic “sensory synapse”.FASEB J.283064–3074. 10.1096/fj.13-245282
26
RobinsonD. R.McNaughtonP. A.EvansM. L.HicksG. A. (2004). Characterization of the primary spinal afferent innervation of the mouse colon using retrograde labelling.Neurogastroenterol. Motil.16113–124. 10.1046/j.1365-2982.2003.00456.x
27
RuF.SurdenikovaL.BrozmanovaM.KollarikM. (2011). Adenosine-induced activation of esophageal nociceptors.Am. J. Physiol. Gastrointest. Liver Physiol.300G485–G493. 10.1152/ajpgi.00361.2010
28
SharradD. F.HibberdT. J.KylohM. A.BrookesS. J.SpencerN. J. (2015). Quantitative immunohistochemical co-localization of TRPV1 and CGRP in varicose axons of the murine oesophagus, stomach and colorectum.Neurosci. Lett.599164–171. 10.1016/j.neulet.2015.05.020
29
SpencerN. J.HuH. (2020). Enteric nervous system: Sensory transduction, neural circuits and gastrointestinal motility.Nat. Rev. Gastroenterol. Hepatol.17338–351. 10.1038/s41575-020-0271-2
30
SpencerN. J.SmithT. K. (2004). Mechanosensory S-neurons rather than AH-neurons appear to generate a rhythmic motor pattern in guinea-pig distal colon.J. Physiol.558(Pt 2)577–596. 10.1113/jphysiol.2004.063586
31
SpencerN. J.GreenheighS.KylohM.HibberdT. J.SharmaH.GrundyL.et al (2018). Identifying unique subtypes of spinal afferent nerve endings within the urinary bladder of mice.J. Comp. Neurol.526707–720. 10.1002/cne.24362
32
SpencerN. J.KerrinA.SingerC. A.HennigG. W.GerthofferW. T.McDonnellO. (2008). Identification of capsaicin-sensitive rectal mechanoreceptors activated by rectal distension in mice.Neuroscience153518–534. 10.1016/j.neuroscience.2008.02.054
33
SpencerN. J.KylohM. A.TravisL.DoddsK. N. (2020a). Identification of spinal afferent nerve endings in the colonic mucosa and submucosa that communicate directly with the spinal cord: The gut-brain axis.J. Comp. Neurol.5281742–1753. 10.1002/cne.24854
34
SpencerN. J.KylohM. A.TravisL.DoddsK. N. (2020b). Sensory nerve endings arising from single spinal afferent neurons that innervate both circular muscle and myenteric ganglia in mouse colon: Colon-brain axis.Cell Tissue Res.38125–34. 10.1007/s00441-020-03192-y
35
SpencerN. J.KylohM.DuffieldM. (2014). Identification of different types of spinal afferent nerve endings that encode noxious and innocuous stimuli in the large intestine using a novel anterograde tracing technique.PLoS One9:e112466. 10.1371/journal.pone.0112466
36
SpencerN. J.KylohM.BeckettE. A.BrookesS.HibberdT. (2016). Different types of spinal afferent nerve endings in stomach and esophagus identified by anterograde tracing from dorsal root ganglia.J. Comp. Neurol.5243064–3083. 10.1002/cne.24006
37
ThaiJ.KylohM.TravisL.SpencerN. J.IvanusicJ. J. (2020). Identifying spinal afferent (sensory) nerve endings that innervate the marrow cavity and periosteum using anterograde tracing.J. Comp. Neurol.5281903–1916. 10.1002/cne.24862
38
ZagorodnyukV. P.KylohM.BrookesS. J.NicholasS. J.SpencerN. J. (2012). Firing patterns and functional roles of different classes of spinal afferents in rectal nerves during colonic migrating motor complexes in mouse colon.Am. J. Physiol. Gastrointest. Liver Physiol.303G404–G411. 10.1152/ajpgi.00047.2012
39
ZagorodnyukV. P.KylohM.NicholasS.PeirisH.BrookesS. J.ChenB. N.et al (2011). Loss of visceral pain following colorectal distension in an endothelin-3 deficient mouse model of Hirschsprung’s disease.J. Physiol.589(Pt 7)1691–1706. 10.1113/jphysiol.2010.202820
Summary
Keywords
pain, nociception, spinal afferent, visceral pain, colon
Citation
Spencer NJ, Hibberd T, Xie Z and Hu H (2022) How should we define a nociceptor in the gut-brain axis?. Front. Neurosci. 16:1096405. doi: 10.3389/fnins.2022.1096405
Received
12 November 2022
Accepted
05 December 2022
Published
19 December 2022
Volume
16 - 2022
Edited by
Vaughan G. Macefield, Baker Heart and Diabetes Institute, Australia
Reviewed by
John Broad, Queen Mary University of London, United Kingdom
Updates
Copyright
© 2022 Spencer, Hibberd, Xie and Hu.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Nick J. Spencer, nicholas.spencer@flinders.edu.au
†These authors share senior authorship
This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Neuroscience
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.