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.
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 Ca
-imaging.
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 Ca
-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation.
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.
The transient receptor potential (subfamily M, member 8; TRPM8) is a nonselective cation channel localized in primary sensory neurons, and is a candidate for cold thermosensing, mediation of cold ...pain, and bladder overactivity. Studies with TRPM8 knockout mice and selective TRPM8 channel blockers demonstrate a lack of cold sensitivity and reduced cold pain in various rodent models. Furthermore, TRPM8 blockers significantly lower body temperature. We have identified a moderately potent (IC50 = 103 nM), selective TRPM8 antagonist, PF-05105679 (R)-3-(1-(4-fluorophenyl)ethyl)(quinolin-3-ylcarbonyl)aminomethylbenzoic acid. It demonstrated activity in vivo in the guinea pig bladder ice water and menthol challenge tests with an IC50 of 200 nM and reduced core body temperature in the rat (at concentrations >1219 nM). PF-05105679 was suitable for acute administration to humans and was evaluated for effects on core body temperature and experimentally induced cold pain, using the cold pressor test. Unbound plasma concentrations greater than the IC50 were achieved with 600- and 900-mg doses. The compound displayed a significant inhibition of pain in the cold pressor test, with efficacy equivalent to oxycodone (20 mg) at 1.5 hours postdose. No effect on core body temperature was observed. An unexpected adverse event (hot feeling) was reported, predominantly periorally, in 23 and 36% of volunteers (600- and 900-mg dose, respectively), which in two volunteers was nontolerable. In conclusion, this study supports a role for TRPM8 in acute cold pain signaling at doses that do not cause hypothermia.
The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine
intestine. Multiunit activity was recorded in vitro from mesenteric ...afferents supplying segments of mouse jejunum taken from wild-type (WT) and TRPV1 knockout (TRPV1 â/â ) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so
the pressureâresponse curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase
in activity. Afferent response to distension was significantly lower in TRPV1 â/â than in WT mice. Single-unit analysis revealed three functional types of afferent fibres: (1) low-threshold fibres (2) wide
dynamic range fibres and (3) high-threshold fibres. There was a marked downward shift of the pressureâresponse curve for wide
dynamic range fibres in the TRPV1 â/â mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 m m ) was also attenuated in the TRPV1 â/â mice. In contrast, the response to bath application of bradykinin (1 μ m , 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μ m ) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous
discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1 â/â afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the
results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.
Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine ...nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(-/-) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.
NaV1.9 regulates normal colonic afferent mechanosensation and is required for hypersensitivity to noxious inflammatory mediators and those derived from inflammatory bowel disease tissues.
Chronic ...visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage-gated sodium channel subtype NaV1.9 is expressed in half of gut-projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9−/− mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus–response function to ramp colonic distension. By contrast, responses to rapid, high-intensity phasic distension of the colon are initially unaffected; however, run-down of responses to repeat phasic distension were exacerbated in NaV1.9−/− afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9−/− mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high-value target for development of visceral analgesics.
Key points
Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability.
Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and ...inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown.
Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling.
These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
Voltage‐gated sodium channel NaV1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor‐specific NaV1.7 knockout mouse (NaV1.7Nav1.8) and selective small‐molecule NaV1.7 antagonist PF‐5198007. NaV1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve–gut preparations in mouse, or following antagonism of NaV1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage‐gated sodium channel α subunits revealed NaV1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV1.7 (in NaV1.8‐expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV1.7 antagonist PF‐5198007. Our data demonstrate that NaV1.7 (in NaV1.8‐expressing neurons) contributes to defined pain pathways in a modality‐dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.
Key points
Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability.
Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown.
Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling.
These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
The ability to sense visceral pain during appendicitis is diminished with age leading to delay in seeking health care and poorer clinical outcomes. To understand the mechanistic basis of this ...phenomenon, we examined visceral nociception in aged mouse and human tissue. Inflamed and noninflamed appendixes were collected from consenting patients undergoing surgery for the treatment of appendicitis or bowel cancer. Supernatants were generated by incubating samples in buffer and used to stimulate multiunit activity in intestinal preparations, or single-unit activity from teased fibres in colonic preparations, of young and old mice. Changes in afferent innervation with age were determined by measuring the density of calcitonin gene-related peptide-positive afferent fibres and by counting dorsal root ganglia back-labelled by injection of tracer dye into the wall of the colon. Finally, the effect of age on nociceptor function was studied in mouse and human colon. Afferent responses to appendicitis supernatants were greatly impaired in old mice. Further investigation revealed this was due to a marked reduction in the afferent innervation of the bowel and a substantial impairment in the ability of the remaining afferent fibres to transduce noxious stimuli. Translational studies in human tissue demonstrated a significant reduction in the multiunit but not the single-unit colonic mesenteric nerve response to capsaicin with age, indicative of a loss of nociceptor innervation. Our data demonstrate that anatomical and functional deficits in nociception occur with age, underpinning the atypical or silent presentation of appendicitis in the elderly.
Preclinical drug development for visceral pain has largely relied on quantifying pseudoaffective responses to colorectal distension (CRD) in restrained rodents. However, the predictive value of ...changes in simple reflex responses in rodents for the complex human pain experience is not known. Male rats were implanted with venous cannulas and with telemetry transmitters for abdominal electromyographic (EMG) recordings. (14)C-iodoantipyrine was injected during noxious CRD (60 mmHg) in the awake, nonrestrained animal. Regional cerebral blood flow (rCBF)-related tissue radioactivity was quantified by autoradiography and analyzed in the three-dimensionally reconstructed brain by statistical parametric mapping. 60-mmHg CRD, compared with controls (0 mmHg) evoked significant increases in EMG activity (267+/-24% vs. 103+/-8%), as well as in behavioral pain score (77+/-6% vs. 3+/-3%). CRD elicited significant increases in rCBF as expected in sensory (insula, somatosensory cortex), and limbic and paralimbic regions (including anterior cingulate cortex and amygdala). Significant decreases in rCBF were seen in the thalamus, parabrachial nucleus, periaqueductal gray, hypothalamus and pons. Correlations of rCBF with EMG and with behavioral pain score were noted in the cingulate, insula, lateral amygdala, dorsal striatum, somatosensory and motor regions. Our findings support the validity of measurements of cerebral perfusion during CRD in the freely moving rat as a model of functional brain changes in human visceral pain. However, not all regions demonstrating significant group differences correlated with EMG or behavioral measures. This suggests that functional brain imaging captures more extensive responses of the central nervous system to noxious visceral distension than those identified by traditional measures.
Studies in healthy human subjects and patients with irritable bowel syndrome suggest sex differences in cerebral nociceptive processing. Here we examine sex differences in functional brain activation ...in the rat during colorectal distention (CRD), a preclinical model of acute visceral pain. (14)C-iodoantipyrine was injected intravenously in awake, non-restrained female rats during 60- or 0-mmHg CRD while electromyographic abdominal activity (EMG) and pain behavior were recorded. Regional cerebral blood flow-related tissue radioactivity was analyzed by statistical parametric mapping from autoradiographic images of three-dimensionally reconstructed brains. Sex differences were addressed by comparing the current data with our previously published data collected from male rats. While sex differences in EMG and pain scores were modest, significant differences were noted in functional brain activation. Females showed widespread changes in limbic (amygdala, hypothalamus) and paralimbic structures (ventral striatum, nucleus accumbens, raphe), while males demonstrated broad cortical changes. Sex differences were apparent in the homeostatic afferent network (parabrachial nucleus, thalamus, insular and dorsal anterior cingulate cortices), in an emotional-arousal network (amygdala, locus coeruleus complex), and in cortical areas modulating these networks (prefrontal cortex). Greater activation of the ventromedial prefrontal cortex and broader limbic/paralimbic changes in females suggest greater engagement of affective mechanisms during visceral pain. Greater cortical activation in males is consistent with the concept of greater cortical inhibitory effects on limbic structures in males, which may relate to differences in attentional and cognitive attribution to visceral stimuli. These findings show remarkable similarities to reported sex differences in brain responses to visceral stimuli in humans.
Somatostatin is an inhibitory peptide present in abundance in the gastrointestinal (GI) tract. The effects of somatostatin
are mediated through its interaction with a family of G-protein-coupled ...receptors, namely sst1â5. Previous evidence suggested
that the sst2 receptor mediates an inhibitory role of somatostatin on GI afferent nerve sensitivity. In the present study
we further evaluated mechanical and chemical sensitivity of mesenteric afferents in mice deficient in the sst2 receptor. Multi-unit
recordings were made from mesenteric afferents from mouse jejunal segments perfused in vitro . Ramp distension of the jejunum up to 60 mmHg induced biphasic increases in afferent activity in both wild-type (WT) and
sst2 gene knock-out (KO) mice. However, the level of afferent activity was significantly higher in the KO ( n
= 15) compared to the WT ( n
= 16) mice across the entire pressure range. The mesenteric afferent sensitivity to acid was evaluated by intraluminal
infusion of hydrochloric acid (HCl 20 m m ) for 2 min. Peak afferent discharge rate following acid infusion was significantly greater in KO (36.76 ± 6.47 impulses s â1 , n
= 7) than in WT preparations (16.53 ± 3.91 impulses s â1 , n
= 5, P < 0.01). The response to bath-applied bradykinin (1 μ m , 3 ml) was not significantly different in the KO and the WT preparations. It is interesting that in the WT preparations,
octreotide inhibited both low- and high-threshold mechanosensory responses, whereas in the sst2 KO group it appeared to inhibit the low-threshold responses preferentially and failed to affect the high-threshold responses.
The results of the present investigation demonstrate that sst2 deficiency is associated with exaggerated jejunal afferent sensitivity to both mechanical and chemical stimulations, suggesting
that somatostatin plays an important inhibitory role in the control of visceral sensitivity by interacting with the sst2 receptor.
PDF
