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Background and Purpose: Tolerance to the analgesic effects of opioids and resultant dose escalation is associated with worsening of side effects and greater addiction risk. Here, we compare the development of tolerance to the conventional opioid fentanyl with a novel pH-sensitive μ-opioid receptor (MOR) agonist, (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP) that is active only in acidic inflammatory microenvironments., Experimental Approach: An opioid tolerance model was developed in male C57BL/6 mice, with and without dextran sulphate sodium colitis, using increasing doses of either fentanyl or NFEPP over 5 days. Visceral nociception was assessed in vivo by measuring visceromotor responses (VMRs) to noxious colorectal distensions and in vitro measuring colonic afferent nerve activity of mesenteric nerves and performing patch-clamp recordings from isolated dorsal root ganglia neurons. Somatic thermal nociception was tested using a tail immersion assay. Cardiorespiratory effects were analysed by pulse oximeter experiments., Key Results: VMRs and tail immersion tests demonstrated tolerance to fentanyl, but not to NFEPP in colitis mice. Cross-tolerance also occurred to fentanyl, but not to NFEPP. The MOR agonist DAMGO inhibited colonic afferent nerve activity in colitis mice exposed to chronic NFEPP, but not those from fentanyl-treated mice. Similarly, in patch-clamp recordings from isolated dorsal root ganglia neurons, DAMGO inhibited neurons from NFEPP-, but not fentanyl-treated mice., Conclusion and Implications: NFEPP did not exhibit tolerance in an inflammatory pain model, unlike fentanyl. Consequently, dose escalation to maintain analgesia during an evolving inflammation could be avoided, mitigating the potential risk of side effects., (© 2024 British Pharmacological Society.)

Abdominal pain is a cardinal symptom of inflammatory bowel disease (IBD). Transient receptor potential (TRP) channels contribute to abdominal pain in preclinical models of IBD, and TRP melastatin 3 (TRPM3) has recently been implicated in inflammatory bladder and joint pain in rodents. We hypothesized that TRPM3 is involved in colonic sensation and is sensitized during colitis. We used immunohistochemistry, ratiometric Ca 2+ imaging, and colonic afferent nerve recordings in mice to evaluate TRPM3 protein expression in colon-projecting dorsal root ganglion (DRG) neurons, as well as functional activity in DRG neurons and colonic afferent nerves. Colitis was induced using dextran sulfate sodium (DSS) in drinking water. TRPM3 protein expression was observed in 76% of colon-projecting DRG neurons and was often colocalized with calcitonin gene-related peptide. The magnitudes of intracellular Ca 2+ transients in DRG neurons in response to the TRPM3 agonists CIM-0216 and pregnenolone sulfate sodium were significantly greater in neurons from mice with colitis compared with controls. In addition, the percentage of DRG neurons from mice with colitis that responded to CIM-0216 was significantly increased. CIM-0216 also increased the firing rate of colonic afferent nerves from control and mice with colitis. The TRPM3 inhibitor isosakuranetin inhibited the mechanosensitive response to distension of wide dynamic range afferent nerve units from mice with colitis but had no effect in control mice. Thus, TRPM3 contributes to colonic sensory transduction and may be a potential target for treating pain in IBD. NEW & NOTEWORTHY This is the first study to characterize TRPM3 protein expression and function in colon-projecting DRG neurons. A TRPM3 agonist excited DRG neurons and colonic afferent nerves from healthy mice. TRPM3 agonist responses in DRG neurons were elevated during colitis. Inhibiting TRPM3 reduced the firing of wide dynamic range afferent nerves from mice with colitis but had no effect in control mice.

Background & Aims: Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons., Methods: In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 μg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition., Results: Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 μmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2., Conclusions: Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Targeting the acidified inflammatory microenvironment with pH-sensitive opioids is a novel approach for managing visceral pain while mitigating side effects. The analgesic efficacy of pH-dependent opioids has not been studied during the evolution of inflammation, where fluctuating tissue pH and repeated therapeutic dosing could influence analgesia and side effects. Whether pH-dependent opioids can inhibit human nociceptors during extracellular acidification is unexplored. We studied the analgesic efficacy and side-effect profile of a pH-sensitive fentanyl analog, (±)- N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide (NFEPP), during the evolution of colitis induced in mice with dextran sulphate sodium. Colitis was characterized by granulocyte infiltration, histological damage, and acidification of the mucosa and submucosa at sites of immune cell infiltration. Changes in nociception were determined by measuring visceromotor responses to noxious colorectal distension in conscious mice. Repeated doses of NFEPP inhibited nociception throughout the course of disease, with maximal efficacy at the peak of inflammation. Fentanyl was antinociceptive regardless of the stage of inflammation. Fentanyl inhibited gastrointestinal transit, blocked defaecation, and induced hypoxemia, whereas NFEPP had no such side effects. In proof-of-principle experiments, NFEPP inhibited mechanically provoked activation of human colonic nociceptors under acidic conditions mimicking the inflamed state. Thus, NFEPP provides analgesia throughout the evolution of colitis with maximal activity at peak inflammation. The actions of NFEPP are restricted to acidified layers of the colon, without common side effects in normal tissues. N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide could provide safe and effective analgesia during acute colitis, such as flares of ulcerative colitis., (Copyright © 2023 International Association for the Study of Pain.)

Background: Monosodium glutamate (MSG) has been identified as a trigger of abdominal pain in irritable bowel syndrome (IBS), but the mechanism is unknown. This study examined whether MSG causes visceral hypersensitivity using a water-avoidance stress (WAS) mouse model of visceral pain., Methods: Mice were divided into four groups receiving treatment for 6 days: WAS + MSG gavage, WAS + saline gavage, sham-WAS + MSG gavage, and sham-WAS + saline gavage. The acute effects of intraluminal administration of 10 μM MSG on jejunal extrinsic afferent nerve sensitivity to distension (0-60 mmHg) were examined using ex vivo extracellular recordings. MSG was also applied directly to jejunal afferents from untreated mice. Glutamate concentration was measured in serum, and in the serosal compartment of Ussing chambers following apical administration., Key Results: Acute intraluminal MSG application increased distension responses of jejunal afferent nerves from mice exposed to WAS + MSG. This effect was mediated by wide dynamic range and high-threshold units at both physiologic and noxious pressures (10-60 mmHg, p < 0.05). No effect of MSG was observed in the other groups, or when applied directly to the jejunal afferent nerves. Serum glutamate was increased in mice exposed to WAS + MSG compared to sham-WAS + saline, and serosal glutamate increased using WAS tissue (p = 0.0433)., Conclusions and Inferences: These findings demonstrate that repeated exposure to MSG in mice leads to sensitization of jejunal afferent nerves to acute ex vivo exposure to MSG. This may contribute to visceral hypersensitivity reported in response to MSG in patients with IBS., (© 2023 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

The gut-brain axis has received increasing attention recently due to evidence that colonic microbes can affect brain function and behavior. However, little is known about the innervation of the colon by a major component of the gut-brain axis, vagal afferent neurons. Furthermore, it is currently unknown whether individual NG neurons or DRG neurons innervate both the proximal and distal colon. We aimed to quantify the number of vagal and spinal afferent neurons that innervate the colon; and determine whether these individual neurons simultaneously innervate the mouse proximal and distal colon. C57Bl/6 mice received injections of a combination of retrograde tracers that were either injected into the muscularis externa of the proximal or the distal colon: fast blue, DiI and DiO. Five to seven percent of lumbosacral and thoracolumbar spinal afferent neurons, and 25% of vagal afferent neurons were labelled by injections of DiI and DiO into the colon. We also found that approximately 8% of NG neurons innervate the distal colon. Ten percent of labeled thoracolumbar and 15% of labeled lumbosacral DRG neurons innervate both the distal and proximal colon. Eighteen percent of labeled NG neurons innervated both the distal and proximal colon. In conclusion, vagal afferent innervation of the distal colon is less extensive than the proximal colon, whereas a similar gradient was not observed for the spinal afferent innervation. Furthermore, overlap appears to exist between the receptive fields of vagal and spinal afferent neurons that innervate the proximal and distal colon., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Proteases are an evolutionarily conserved family of enzymes that degrade peptide bonds and have been implicated in several common gastrointestinal (GI) diseases. Although luminal proteolytic activity is important for maintenance of homeostasis and health, the current review describes recent advances in our understanding of how overactivity of luminal proteases contributes to the pathophysiology of celiac disease, irritable bowel syndrome, inflammatory bowel disease and GI infections. Luminal proteases, many of which are produced by the microbiota, can modulate the immunogenicity of dietary antigens, reduce mucosal barrier function and activate pro-inflammatory and pro-nociceptive host signaling. Increased proteolytic activity has been ascribed to both increases in protease production and decreases in inhibitors of luminal proteases. With the identification of strains of bacteria that are important sources of proteases and their inhibitors, the stage is set to develop drug or microbial therapies to restore protease balance and alleviate disease.

Objective: Dietary therapies for irritable bowel syndrome (IBS) have received increasing interest but predicting which patients will benefit remains a challenge due to a lack of mechanistic insight. We recently found evidence of a role for the microbiota in dietary modulation of pain signalling in a humanised mouse model of IBS. This randomised cross-over study aimed to test the hypothesis that pain relief following reduced consumption of fermentable carbohydrates is the result of changes in luminal neuroactive metabolites., Design: IBS (Rome IV) participants underwent four trial periods: two non-intervention periods, followed by a diet low (LFD) and high in fermentable carbohydrates for 3 weeks each. At the end of each period, participants completed questionnaires and provided stool. The effects of faecal supernatants (FS) collected before (IBS FS) and after a LFD (LFD FS) on nociceptive afferent neurons were assessed in mice using patch-clamp and ex vivo colonic afferent nerve recording techniques., Results: Total IBS symptom severity score and abdominal pain were reduced by the LFD (N=25; p<0.01). Excitability of neurons was increased in response to IBS FS, but this effect was reduced (p<0.01) with LFD FS from pain-responders. IBS FS from pain-responders increased mechanosensitivity of nociceptive afferent nerve axons (p<0.001), an effect lost following LFD FS administration (p=NS) or when IBS FS was administered in the presence of antagonists of histamine receptors or protease inhibitors., Conclusions: In a subset of IBS patients with improvement in abdominal pain following a LFD, there is a decrease in pronociceptive signalling from FS, suggesting that changes in luminal mediators may contribute to symptom response., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Endometriosis patients experience debilitating chronic pain, and the first-line treatment is ineffective at managing symptoms. Although surgical removal of the lesions provides temporary relief, more than 50% of the patients experience disease recurrence. Despite being a leading cause of hysterectomy, endometriosis lacks satisfactory treatments and a cure. Another challenge is the poor understanding of disease pathophysiology which adds to the delays in diagnosis and overall compromised quality of life. Endometriosis patients are in dire need of an effective therapeutic strategy that is both economical and effective in managing symptoms, while fertility is unaffected. Endocannabinoids and phytocannabinoids possess anti-inflammatory, anti-nociceptive, and anti-proliferative properties that may prove beneficial for endometriosis management, given that inflammation, vascularization, and pain are hallmark features of endometriosis. Endocannabinoids are a complex network of molecules that play a central role in physiological processes including homeostasis and tissue repair, but endocannabinoids have also been associated in the pathophysiology of several chronic inflammatory diseases including endometriosis and cancers. The lack of satisfactory treatment options combined with the recent legalization of recreational cannabinoids in some parts of the world has led to a rise in self-management strategies including the use of cannabinoids for endometriosis-related pain and other symptoms. In this review, we provide a comprehensive overview of endocannabinoids with a focus on their potential roles in the pathophysiology of endometriosis. We further provide evidence-driven perspectives on the current state of knowledge on endometriosis-associated pain, inflammation, and therapeutic avenues exploiting the endocannabinoid system for its management., (© 2022. The Author(s).)

While effective in treating abdominal pain, opioids have significant side effects. Recent legalization of cannabis will likely promote use of cannabinoids as an adjunct or alternative to opioids, despite a lack of evidence. We aimed to investigate whether cannabinoids inhibit mouse colonic nociception, alone or in combination with opioids at low doses. Experiments were performed on C57BL/6 male and female mice. Visceral nociception was evaluated by measuring visceromotor responses (VMR), afferent nerve mechanosensitivity in flat-sheet colon preparations, and excitability of isolated DRG neurons. Blood oxygen saturation, locomotion, and defecation were measured to evaluate side effects. An agonist of cannabinoid 1 receptor (CB1R), arachidonyl-2'-chloroethylamide (ACEA), dose-dependently decreased VMR. ACEA and HU-210 (another CB1R agonist) also attenuated colonic afferent nerve mechanosensitivity. Additionally, HU-210 concentration-dependently decreased DRG neuron excitability, which was reversed by the CB1R antagonist AM-251. Conversely, cannabinoid 2 receptor (CB2R) agonists did not attenuate VMR, afferent nerve mechanosensitivity, or DRG neuron excitability. Combination of subanalgesic doses of CB1R and µ-opioid receptor agonists decreased VMR; importantly, this analgesic effect was preserved after 6 d of twice daily treatment. This combination also attenuated afferent nerve mechanosensitivity and DRG neuron excitability, which was inhibited by neuronal nitric oxide synthase and guanylate cyclase inhibitors. This combination avoided side effects (decreased oxygen saturation and colonic transit) caused by analgesic dose of morphine. Activation of CB1R, but not CB2R, decreased colonic nociception both alone and in synergy with µ-opioid receptor. Thus, CB1R agonists may enable opioid dose reduction and avoid opioid-related side effects. SIGNIFICANCE STATEMENT One of the most cited needs for patients with abdominal pain are safe and effective treatment options. The effectiveness of opioids in the management of abdominal pain is undermined by severe adverse side effects. Therefore, strategies to replace opioids or reduce the doses of opioids to suppress abdominal pain is needed. This study in mice demonstrates that cannabinoid 1 receptor (CB1R) agonists inhibit visceral sensation. Furthermore, a combination of subanalgesic doses of µ-opioid receptor agonist and CB1R agonist markedly reduce abdominal pain without causing the side effects of high-dose opioids. Thus, CB1R agonists, alone or in combination with low-dose opioids, may be a novel and safe treatment strategy for abdominal pain., (Copyright © 2022 the authors.)

The gut microbiota has been implicated in chronic pain disorders, including irritable bowel syndrome (IBS), yet specific pathophysiological mechanisms remain unclear. We showed that decreasing intake of fermentable carbohydrates improved abdominal pain in patients with IBS, and this was accompanied by changes in the gut microbiota and decreased urinary histamine concentrations. Here, we used germ-free mice colonized with fecal microbiota from patients with IBS to investigate the role of gut bacteria and the neuroactive mediator histamine in visceral hypersensitivity. Germ-free mice colonized with the fecal microbiota of patients with IBS who had high but not low urinary histamine developed visceral hyperalgesia and mast cell activation. When these mice were fed a diet with reduced fermentable carbohydrates, the animals showed a decrease in visceral hypersensitivity and mast cell accumulation in the colon. We observed that the fecal microbiota from patients with IBS with high but not low urinary histamine produced large amounts of histamine in vitro. We identified Klebsiella aerogenes , carrying a histidine decarboxylase gene variant, as a major producer of this histamine. This bacterial strain was highly abundant in the fecal microbiota of three independent cohorts of patients with IBS compared with healthy individuals. Pharmacological blockade of the histamine 4 receptor in vivo inhibited visceral hypersensitivity and decreased mast cell accumulation in the colon of germ-free mice colonized with the high histamine-producing IBS fecal microbiota. These results suggest that therapeutic strategies directed against bacterial histamine could help treat visceral hyperalgesia in a subset of patients with IBS with chronic abdominal pain.

Opioid tolerance (OT) leads to dose escalation and serious side effects, including opioid-induced hyperalgesia (OIH). We sought to better understand the mechanisms underlying this event in the gastrointestinal tract. Chronic in vivo administration of morphine by intraperitoneal injection in male C57BL/6 mice evoked tolerance and evidence of OIH in an assay of colonic afferent nerve mechanosensitivity; this was inhibited by the δ-opioid receptor (DOPr) antagonist naltrindole when intraperitoneally injected in previous morphine administration. Patch-clamp studies of DRG neurons following overnight incubation with high concentrations of morphine, the µ-opioid receptors (MOPr) agonist [D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-Enkephalin (DAMGO) or the DOPr agonist [D-Ala 2 , D-Leu 5 ]-Enkephalin evoked hyperexcitability. The pronociceptive actions of these opioids were blocked by the DOPr antagonist SDM25N but not the MOPr antagonist D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH 2 The hyperexcitability induced by DAMGO was reversed after a 1 h washout, but reapplication of low concentrations of DAMGO or [D-Ala 2 , D-Leu 5 ]-Enkephalin restored the hyperexcitability, an effect mediated by protein kinase C. DOPr-dependent DRG neuron hyperexcitability was blocked by the endocytosis inhibitor Pitstop 2, and the weakly internalizing DOPr agonist ARM390 did not cause hyperexcitability. Bioluminescence resonance energy transfer studies in HEK cells showed no evidence of switching of G-protein signaling from G i to a G s pathway in response to either high concentrations or overnight incubation of opioids. Thus, chronic high-dose opioid exposure leads to opioid tolerance and features of OIH in the colon. This action is mediated by DOPr signaling and is dependent on receptor endocytosis and downstream protein kinase C signaling. SIGNIFICANCE STATEMENT Opioids are effective in the treatment of abdominal pain, but escalating doses can lead to opioid tolerance and potentially opioid-induced hyperalgesia. We found that δ-opioid receptor (DOPr) plays a central role in the development of opioid tolerance and opioid-induced hyperalgesia in colonic afferent nociceptors following prolonged exposure to high concentrations of MOPr or DOPr agonists. Furthermore, the role of DOPr was dependent on OPr internalization and activation of a protein kinase C signaling pathway. Thus, targeting DOPr or key components of the downstream signaling pathway could mitigate adverse side effects by opioids., (Copyright © 2022 the authors.)

Objective: The effectiveness of µ-opioid receptor (MOPr) agonists for treatment of visceral pain is compromised by constipation, respiratory depression, sedation and addiction. We investigated whether a fentanyl analogue, (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP), which preferentially activates MOPr in acidified diseased tissues, would inhibit pain in a preclinical model of inflammatory bowel disease (IBD) without side effects in healthy tissues., Design: Antinociceptive actions of NFEPP and fentanyl were compared in control mice and mice with dextran sodium sulfate colitis by measuring visceromotor responses to colorectal distension. Patch clamp and extracellular recordings were used to assess nociceptor activation. Defecation, respiration and locomotion were assessed. Colonic migrating motor complexes were assessed by spatiotemporal mapping of isolated tissue. NFEPP-induced MOPr signalling and trafficking were studied in human embryonic kidney 293 cells., Results: NFEPP inhibited visceromotor responses to colorectal distension in mice with colitis but not in control mice, consistent with acidification of the inflamed colon. Fentanyl inhibited responses in both groups. NFEPP inhibited the excitability of dorsal root ganglion neurons and suppressed mechanical sensitivity of colonic afferent fibres in acidified but not physiological conditions. Whereas fentanyl decreased defecation and caused respiratory depression and hyperactivity in mice with colitis, NFEPP was devoid of these effects. NFEPP did not affect colonic migrating motor complexes at physiological pH. NFEPP preferentially activated MOPr in acidified extracellular conditions to inhibit cAMP formation, recruit β-arrestins and evoke MOPr endocytosis., Conclusion: In a preclinical IBD model, NFEPP preferentially activates MOPr in acidified microenvironments of inflamed tissues to induce antinociception without causing respiratory depression, constipation and hyperactivity., Competing Interests: Competing interests: NWB is a founding scientist of Endosome Therapeutics Inc., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Endometriosis (EM) is characterized by the growth of endometrium-like tissue outside the uterus, leading to chronic inflammation and pelvic pain. Lesion proliferation, vascularization, and associated inflammation are the hallmark features of EM lesions. The legalization of recreational cannabinoids has garnered interest in the patient community and is contributing to a greater incidence of self medication; however, it remains unknown if cannabinoids possess marked disease-modifying properties. In this study, we assess the effects of synthetic cannabinoid, WIN 55212-2 (WIN 55), in EM-representative in vitro and in vivo syngeneic mouse models. WIN 55 reduced proliferation and angiogenesis in vitro, via MAPK/Akt-mediated apoptosis. These findings were corroborated in a mouse model of EM, where we found reduced TRPV1 expression in the dorsal root ganglia of the EM mouse model exposed to WIN 55, suggesting reduced signaling of pain stimuli. Ultimately, these pieces of evidence support the use of cannabinoid receptor agonists as a potential therapeutic intervention for EM associated pain and inflammation., Competing Interests: 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., (Copyright © 2021 Lingegowda, Miller, Marks, Symons, Alward, Lomax, Koti and Tayade.)

Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gα i/o and β-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain., Competing Interests: Competing interest statement: N.W.B. is a founding scientist of Endosome Therapeutics Inc. Research in the laboratories of N.A.V., N.W.B., and D.P.P. is funded in part by Takeda Pharmaceuticals International.

Key Points: The vagus nerve has been implicated in mediating behavioural effects of the gut microbiota on the central nervous system. This study examined whether the secretory products of commensal gut bacteria can modulate the excitability of vagal afferent neurons with cell bodies in nodose ganglia. Cysteine proteases from commensal bacteria increased the excitability of vagal afferent neurons via activation of protease-activated receptor 2 and modulation of the voltage dependence of Na + conductance activation. Lipopolysaccharide, a component of the cell wall of gram-negative bacteria, increased the excitability of nodose ganglia neurons via TLR4-dependent activation of nuclear factor kappa B. Our study identified potential mechanisms by which gut microbiota influences the activity of vagal afferent pathways, which may in turn impact on autonomic reflexes and behaviour., Abstract: Behavioural studies have implicated vagal afferent neurons as an important component of the microbiota-gut-brain axis. However, the mechanisms underlying the ability of the gut microbiota to affect vagal afferent pathways are unclear. We examined the effect of supernatant from a community of 33 commensal gastrointestinal bacterial derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) on the excitability of mouse vagal afferent neurons. Perforated patch clamp electrophysiology was used to measure the excitability of dissociated nodose ganglion (NG) neurons. NG neuronal excitability was assayed by measuring the amount of current required to elicit an action potential, the rheobase. MET-1 supernatant increased the excitability of NG neurons by hyperpolarizing the voltage dependence of activation of Na + conductance. The increase in excitability elicited by MET-1 supernatant was blocked by the cysteine protease inhibitor E-64 (30 nm). The protease activated receptor-2 (PAR 2 ) antagonist (GB 83, 10 μm) also blocked the effect of MET-1 supernatant on NG neurons. Supernatant from Lactobacillus paracasei 6MRS, a component of MET-1, recapitulated the effect of MET-1 supernatant on NG neurons. Lastly, we compared the effects of MET-1 supernatant and lipopolysaccharide (LPS) from Escherichia coli 05:B5 on NG neuron excitability. LPS increased the excitability of NG neurons in a toll-like receptor 4 (TLR 4 )-dependent and PAR 2 -independent manner, whereas the excitatory effects of MET-1 supernatant were independent of TLR 4 activation. Together, our findings suggest that cysteine proteases from commensal bacteria increase the excitability of vagal afferent neurons by the activation of PAR 2 ., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Background: A low fermentable carbohydrate (FODMAP) diet is used in quiescent inflammatory bowel disease when irritable bowel syndrome-like symptoms occur. There is concern that the diet could exacerbate inflammation by modifying microbiota and short-chain fatty acid (SCFA) production. We examined the effect of altering dietary FODMAP content on inflammation in preclinical inflammatory models., Methods: C57BL/6 mice were given 3% dextran sodium sulfate (DSS) in drinking water for 5 days and recovered for 3 weeks (postinflammatory, n = 12), or 5 days (positive-control, n = 12). Following recovery, DSS-treated or control mice (negative-control, n = 12) were randomized to 2-week low- (0.51 g/100 g total FODMAP) or high-FODMAP (4.10 g) diets. Diets mimicked human consumption containing fructose, sorbitol, galacto-oligosaccharide, and fructan. Colons were assessed for myeloperoxidase (MPO) activity and histological damage. Supernatants were generated for perforated patch-clamp recordings and cytokine measurement. Cecum contents were analyzed for microbiota, SCFA, and branched-chain fatty acids (BCFA). Data were analyzed by two-way ANOVA with Bonferroni., Key Results: Inflammatory markers were higher in the positive-control compared with negative-control and postinflammatory groups, but no differences occurred between the two diets within each treatment (MPO P > .99, histological scores P > .99, cytokines P > .05), or the perforated patch-clamp recordings (P > .05). Microbiota clustered mainly based on DSS exposure. No difference in SCFA content occurred. Higher total BCFA occurred with the low-FODMAP diet in positive-control (P < .01) and postinflammatory groups (P < .01)., Conclusions and Inferences: In this preclinical study, reducing dietary FODMAPs did not exacerbate nor mitigate inflammation. Microbiota profile changes were largely driven by inflammation rather than diet. Low FODMAP intake caused a shift toward proteolytic fermentation following inflammation., (© 2019 John Wiley & Sons Ltd.)

The potential role of the intestinal microbiota in modulating visceral pain has received increasing attention during recent years. This has led to the identification of signaling pathways that have been implicated in communication between gut bacteria and peripheral pain pathways. In addition to the well-characterized impact of the microbiota on the immune system, which in turn affects nociceptor excitability, bacteria can modulate visceral afferent pathways by effects on enterocytes, enteroendocrine cells, and the neurons themselves. Proteases produced by bacteria, or by host cells in response to bacteria, can increase or decrease the excitability of nociceptive dorsal root ganglion (DRG) neurons depending on the receptor activated. Short-chain fatty acids generated by colonic bacteria are involved in gut-brain communication, and intracolonic short-chain fatty acids have pronociceptive effects in rodents but may be antinociceptive in humans. Gut bacteria modulate the synthesis and release of enteroendocrine cell mediators, including serotonin and glucagon-like peptide-1, which activate extrinsic afferent neurons. Deciphering the complex interactions between visceral afferent neurons and the gut microbiota may lead to the development of improved probiotic therapies for visceral pain.

Increased bile acids in the colon can evoke increased epithelial secretion resulting in diarrhea, but little is known about whether colonic bile acids contribute to abdominal pain. This study aimed to investigate the mechanisms underlying activation of colonic extrinsic afferent nerves and their neuronal cell bodies by a major secondary bile acid, deoxycholic acid (DCA). All experiments were performed on male C57BL/6 mice. Afferent sensitivity was evaluated using in vitro extracellular recordings from mesenteric nerves in the proximal colon (innervated by vagal and spinal afferents) and distal colon (spinal afferents only). Neuronal excitability of cultured dorsal root ganglion (DRG) and nodose ganglion (NG) neurons was examined with perforated patch clamp. Colonic 5-HT release was assessed using ELISA, and 5-HT immunoreactive enterochromaffin (EC) cells were quantified. Intraluminal DCA increased afferent nerve firing rate concentration dependently in both proximal and distal colon. This DCA-elicited increase was significantly inhibited by a 5-HT 3 antagonist in the proximal colon but not in the distal colon, which may be in part due to lower 5-HT immunoreactive EC cell density and lower 5-HT levels in the distal colon following DCA stimulation. DCA increased the excitability of DRG neurons, whereas it decreased the excitability of NG neurons. DCA potentiated mechanosensitivity of high-threshold spinal afferents independent of 5-HT release. Together, this study suggests that DCA can excite colonic afferents via direct and indirect mechanisms but the predominant mechanism may differ between vagal and spinal afferents. Furthermore, DCA increased mechanosensitivity of high-threshold spinal afferents and may be a mechanism of visceral hypersensitivity. NEW & NOTEWORTHY Deoxycholic acid (DCA) directly excites spinal afferents and, to a lesser extent, indirectly via mucosal 5-HT release. DCA potentiates mechanosensitivity of high-threshold spinal afferents independent of 5-HT release. DCA increases vagal afferent firing in proximal colon via 5-HT release but directly inhibits the excitability of their cell bodies.

We have previously demonstrated that satiety sensing vagal afferent neurons are less responsive to meal-related stimuli in obesity because of reduced electrical excitability. As leak K + currents are key determinants of membrane excitability, we hypothesized that leak K + currents are increased in vagal afferents during obesity. Diet-induced obesity was induced by feeding C57Bl/6J mice a high-fat diet (HFF) (60% energy from fat) for 8-10 wk. In vitro extracellular recordings were performed on jejunal afferent nerves. Whole cell patch-clamp recordings were performed on mouse nodose ganglion neurons. Leak K + currents were isolated using ion substitution and pharmacological blockers. mRNA for TWIK-related acid-sensitive K + (TASK) subunits was measured using quantitative real-time PCR. Intestinal afferent responses to nutrient (oleate) and non-nutrient (ATP) stimuli were significantly decreased in HFF mice. Voltage clamp experiments revealed the presence of a voltage-insensitive resting potassium conductance that was increased by external alkaline pH and halothane, known properties of TASK currents. In HFF neurons, leak K + current was approximately doubled and was reduced by TASK1 and TASK3 inhibitors. The halothane sensitive current was similarly increased. Quantitative PCR revealed the presence of mRNA encoding TASK1 (KCNK3) and TASK3 (KCNK9) channels in nodose neurons. TASK3 transcript was significantly increased in HFF mice. The reduction in vagal afferent excitability in obesity is due in part to an increase of resting (leak) K + conductance. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a therapeutic target for obesity treatment. NEW & NOTEWORTHY This study characterized the electrophysiological properties and gene expression of the TWIK-related acid-sensitive K + (TASK) channel in vagal afferent neurons. TASK conductance was increased and contributed to decreased excitability in diet-induced obesity. TASK channels may account for the impairment of satiety signaling in diet-induced obesity and thus is a promising therapeutic target.

The immune and nervous systems are tightly integrated, with each system capable of influencing the other to respond to infectious or inflammatory perturbations of homeostasis. Recent studies demonstrating the ability of neural stimulation to significantly reduce the severity of immunopathology and consequently reduce mortality have led to a resurgence in the field of neuroimmunology. Highlighting the tight integration of the nervous and immune systems, afferent neurons can be activated by a diverse range of substances from bacterial-derived products to cytokines released by host cells. While activation of vagal afferents by these substances dominates the literature, additional sensory neurons are responsive as well. It is becoming increasingly clear that although the cholinergic anti-inflammatory pathway has become the predominant model, a multitude of functional circuits exist through which neuronal messengers can influence immunological outcomes. These include pathways whereby efferent signaling occurs independent of the vagus nerve through sympathetic neurons. To receive input from the nervous system, immune cells including B and T cells, macrophages, and professional antigen presenting cells express specific neurotransmitter receptors that affect immune cell function. Specialized immune cell populations not only express neurotransmitter receptors, but express the enzymatic machinery required to produce neurotransmitters, such as acetylcholine, allowing them to act as signaling intermediaries. Although elegant experiments have begun to decipher some of these interactions, integration of these molecules, cells, and anatomy into defined neuroimmune circuits in health and disease is in its infancy. This review describes these circuits and highlights continued challenges and opportunities for the field.

Background and Purpose: To better understand opioid signalling in visceral nociceptors, we examined the expression and selective activation of μ and δ opioid receptors by dorsal root ganglia (DRG) neurons innervating the mouse colon., Experimental Approach: DRG neurons projecting to the colon were identified by retrograde tracing. δ receptor-GFP reporter mice, in situ hybridization, single-cell RT-PCR and μ receptor-specific antibodies were used to characterize expression of μ and δ receptors. Voltage-gated Ca 2+ currents and neuronal excitability were recorded in small diameter nociceptive neurons (capacitance <30 pF) by patch clamp and ex vivo single-unit afferent recordings were obtained from the colon., Key Results: In situ hybridization of oprm1 expression in Fast Blue-labelled DRG neurons was observed in 61% of neurons. μ and δ receptors were expressed by 36-46% of colon DRG neurons, and co-expressed by ~25% of neurons. μ and δ receptor agonists inhibited Ca 2+ currents in DRG, effects blocked by opioid antagonists. One or both agonists inhibited action potential firing by colonic afferent endings. Incubation of neurons with supernatants from inflamed colon segments inhibited Ca 2+ currents and neuronal excitability. Antagonists of μ, but not δ receptors, inhibited the effects of these supernatant on Ca 2+ currents, whereas both antagonists inhibited their actions on neuronal excitability., Conclusions and Implications: A significant number of small diameter colonic nociceptors co-express μ and δ receptors and are inhibited by agonists and endogenous opioids in inflamed tissues. Thus, opioids that act at μ or δ receptors, or their heterodimers may be effective in treating visceral pain., (© 2018 The British Pharmacological Society.)

Aims and Background: Psychological stress accompanies chronic inflammatory diseases such as IBD, and stress hormones can exacerbate pain signalling. In contrast, the endogenous opioid system has an important analgesic action during chronic inflammation. This study examined the interaction of these pathways., Methods: Mouse nociceptive dorsal root ganglia (DRG) neurons were incubated with supernatants from segments of inflamed colon collected from patients with chronic UC and mice with dextran sodium sulfate (cDSS)-induced chronic colitis. Stress effects were studied by adding stress hormones (epinephrine and corticosterone) to dissociated neurons or by exposing cDSS mice to water avoidance stress. Changes in excitability of colonic DRG nociceptors were measured using patch clamp and Ca 2+ imaging techniques., Results: Supernatants from patients with chronic UC and from colons of mice with chronic colitis caused a naloxone-sensitive inhibition of neuronal excitability and capsaicin-evoked Ca 2+ responses. Stress hormones decreased signalling induced by human and mouse supernatants. This effect resulted from stress hormones signalling directly to DRG neurons and indirectly through signalling to the immune system, leading to decreased opioid levels and increased acute inflammation. The net effect of stress was a change endogenous opioid signalling in DRG neurons from an inhibitory to an excitatory effect. This switch was associated with a change in G protein-coupled receptor excitatory signalling to a pathway sensitive to inhibitors of protein kinase A-protein, phospholipase C-protein and G protein βϒ subunits., Conclusions: Stress hormones block the inhibitory actions of endogenous opioids and can change the effect of opioid signalling in DRG neurons to excitation. Targeting these pathways may prevent heavy opioid use in IBD., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human donor signal to DRG neurons. Their secretory products contain serine proteases that suppress excitability via activation of protease-activated receptor-4. Moreover, from this community of commensal microbes, Faecalibacterium prausnitzii strain 16-6-I 40 fastidious anaerobe agar had the greatest effect. Our study suggests that therapies that induce or correct microbial dysbiosis may affect the excitability of primary afferent neurons, many of which are nociceptive. Furthermore, identification of the bacterial strains capable of suppressing sensory neuron excitability, and their mechanisms of action, may allow therapeutic relief for patients with gastrointestinal diseases associated with pain., (Copyright © 2017 the authors 0270-6474/17/3711758-11$15.00/0.)

Background: Changes to the structure and function of the innervation of the gut contribute to symptom generation in inflammatory bowel diseases (IBD). However, delineation of the mechanisms of these effects has proven difficult. Previous work on sympathetic neurons identified interleukin (IL)-17A as a novel neurotrophic cytokine. Since IL-17A is involved in IBD pathogenesis, we tested the hypothesis that IL-17A contributes to neuroanatomical remodeling during IBD., Methods: Immunohistochemistry for tyrosine hydroxylase was used to identify sympathetic axons in mice with dextran sulphate sodium (DSS)-induced colitis and controls. Axon outgrowth from sympathetic neurons in response to incubation in cytokines or endoscopic patient biopsy supernatants was quantified., Key Results: DSS-induced colitis led to an increase in tyrosine hydroxylase immunoreactivity in the inflamed colon but not the spleen. Colonic supernatants from mice with colitis and biopsy supernatants from Crohn's disease patients increased axon outgrowth from mouse sympathetic neurons compared to supernatants from uninflamed controls. An antibody that neutralized IL-17A blocked the ability of DSS-induced colitis and Crohn's disease supernatants to induce axon extension., Conclusions and Inferences: These findings identify IL-17A as a potential mediator of neuroanatomical remodeling of the gut innervation during IBD., (© 2017 John Wiley & Sons Ltd.)

Background & Aims: Cell therapy offers the potential to treat gastrointestinal motility disorders caused by diseased or absent enteric neurons. We examined whether neurons generated from transplanted enteric neural cells provide a functional innervation of bowel smooth muscle in mice., Methods: Enteric neural cells expressing the light-sensitive ion channel, channelrhodopsin, were isolated from the fetal or postnatal mouse bowel and transplanted into the distal colon of 3- to 4-week-old wild-type recipient mice. Intracellular electrophysiological recordings of responses to light stimulation of the transplanted cells were made from colonic smooth muscle cells in recipient mice. Electrical stimulation of endogenous enteric neurons was used as a control., Results: The axons of graft-derived neurons formed a plexus in the circular muscle layer. Selective stimulation of graft-derived cells by light resulted in excitatory and inhibitory junction potentials, the electrical events underlying contraction and relaxation, respectively, in colonic muscle cells. Graft-derived excitatory and inhibitory motor neurons released the same neurotransmitters as endogenous motor neurons-acetylcholine and a combination of adenosine triphosphate and nitric oxide, respectively. Graft-derived neurons also included interneurons that provided synaptic inputs to motor neurons, but the pharmacologic properties of interneurons varied with the age of the donors from which enteric neural cells were obtained., Conclusions: Enteric neural cells transplanted into the bowel give rise to multiple functional types of neurons that integrate and provide a functional innervation of the smooth muscle of the bowel wall. Circuits composed of both motor neurons and interneurons were established, but the age at which cells are isolated influences the neurotransmitter phenotype of interneurons that are generated., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Altered gastrointestinal (GI) function contributes to the debilitating symptoms of inflammatory bowel diseases (IBD). Nerve circuits contained within the gut wall and outside of the gut play important roles in modulating motility, mucosal fluid transport, and blood flow. The structure and function of these neuronal populations change during IBD. Superimposed on this plasticity is a diminished responsiveness of effector cells - smooth muscle cells, enterocytes, and vascular endothelial cells - to neurotransmitters. The net result is a breakdown in the precisely orchestrated coordination of motility, fluid secretion, and GI blood flow required for health. In this review, we consider how inflammation-induced changes to the effector innervation of these tissues, and changes to the tissues themselves, contribute to defective GI function in models of IBD. We also explore the evidence that reversing neuronal plasticity is sufficient to normalize function during IBD., (Copyright © 2017 the American Physiological Society.)

Changes in diet are a challenge to the gastrointestinal tract which needs to alter its processing mechanisms to continue to process nutrients and maintain health. In particular, the enteric nervous system (ENS) needs to adapt its motor and secretory programs to deal with changes in nutrient type and load in order to optimise nutrient absorption.The nerve circuits in the gut are complex, and the numbers and types of neurons make recordings of specific cell types difficult, time-consuming, and prone to sampling errors. Nonetheless, traditional research methods like intracellular electrophysiological approaches have provided the basis for our understanding of the ENS circuitry. In particular, animal models of intestinal inflammation have shown us that we can document changes to neuronal excitability and synaptic transmission.Recent studies examining diet-induced changes to ENS programming have opted to use fast imaging techniques to reveal changes in neuron function. Advances in imaging techniques using voltage- or calcium-sensitive dyes to record neuronal activity promise to overcome many limitations inherent to electrophysiological approaches. Imaging techniques allow access to a wide range of ENS phenotypes and to the changes they undergo during dietary challenges. These sorts of studies have shown that dietary variation or obesity can change how the ENS processes information-in effect reprogramming the ENS. In this review, the data gathered from intracellular recordings will be compared with measurements made using imaging techniques in an effort to determine if the lessons learnt from inflammatory changes are relevant to the understanding of diet-induced reprogramming.

Constipation arises from a multitude of causes, including aging, spinal cord injury (SCI), and dietary issues. The heterogeneity of inciting factors has made the treatment of constipation particularly challenging. Agonists of ghrelin receptors have beneficial effects on delayed gastric emptying, but less is known about their ability to improve colorectal motility. Recent publications indicate that the activation of the ghrelin receptors in the spinal cord can alleviate constipation due to dietary causes, Parkinsonism, and SCI in rodents. Ghrelin-responsive neurons in the intermediolateral cell column of the lumbosacral spinal cord can activate enteric microcircuits that coordinate propulsive colorectal contractions, leading to defecation. Learning more about the properties of neurons in the spinal defecation center and the roles of ghrelin receptors in the defecation reflex will accelerate the development of improved treatments of constipation., (© 2015 John Wiley & Sons Ltd.)

The enteric nervous system arises from neural crest-derived cells (ENCCs) that migrate caudally along the embryonic gut. The expression of ion channels by ENCCs in embryonic mice was investigated using a PCR-based array, RT-PCR and immunohistochemistry. Many ion channels, including chloride, calcium, potassium and sodium channels were already expressed by ENCCs at E11.5. There was an increase in the expression of numerous ion channel genes between E11.5 and E14.5, which coincides with ENCC migration and the first extension of neurites by enteric neurons. Previous studies have shown that a variety of ion channels regulates neurite extension and migration of many cell types. Pharmacological inhibition of a range of chloride or calcium channels had no effect on ENCC migration in cultured explants or neuritogenesis in vitro. The non-selective potassium channel inhibitors, TEA and 4-AP, retarded ENCC migration and neuritogenesis, but only at concentrations that also resulted in cell death. In summary, a large range of ion channels is expressed while ENCCs are colonizing the gut, but we found no evidence that ENCC migration or neuritogenesis requires chloride, calcium or potassium channel activity. Many of the ion channels are likely to be involved in the development of electrical excitability of enteric neurons.

Sepsis is a severe systemic inflammatory disorder that rapidly activates the sympathetic nervous system to enhance catecholamine secretion from postganglionic sympathetic neurons and adrenal chromaffin cells. Although an increase in preganglionic drive to postganglionic sympathetic tissues has been known to contribute to this response for quite some time, only recently was it determined that sepsis also has direct effects on adrenal chromaffin cell Ca2+ signaling and epinephrine release. In the present study, we characterized the direct effects of sepsis on postganglionic sympathetic neuron function. Using the endotoxemia model of sepsis in mice, we found that almost a quarter of postganglionic neurons acquired the ability to fire spontaneous action potentials, which was absent in cells from control mice. Spontaneously firing neurons possessed significantly lower rheobases and fired a greater number of action potentials at twice the rheobase compared to neurons from control mice. Sepsis did not significantly affect voltage-gated Ca2+ currents. However, global Ca2+ signaling was enhanced in postganglionic neurons isolated from 1 to 24 h endotoxemic mice. A similar increase in the amplitude of high-K+-stimulated Ca2+ transients was observed during the cecal ligation and puncture model of sepsis. The enhanced excitability and Ca2+ signaling produced during sepsis likely amplify the effect of increased preganglionic drive on norepinephrine release from postganglionic neurons. This is important, as sympathetic neurons are integral to the anti-inflammatory autonomic reflex that is activated during sepsis., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

The sympathetic nervous system (SNS) is part of an integrative network that functions to restore homeostasis following injury and infection. The SNS can provide negative feedback control over inflammation through the secretion of catecholamines from postganglionic sympathetic neurons and adrenal chromaffin cells (ACCs). Central autonomic structures receive information regarding the inflammatory status of the body and reflexively modulate SNS activity. However, inflammation and infection can also directly regulate SNS function by peripheral actions on postganglionic cells. The present review discusses how inflammation activates autonomic reflex pathways and compares the effect of localized and systemic inflammation on ACCs and postganglionic sympathetic neurons. Systemic inflammation significantly enhanced catecholamine secretion through an increase in Ca(2+) release from the endoplasmic reticulum. In contrast, acute and chronic GI inflammation reduced voltage-gated Ca(2+) current. Thus it appears that the mechanisms underlying the effects of peripheral and systemic inflammation neuroendocrine function converge on the modulation of intracellular Ca(2+) signaling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Inflammation involving the helper T cell 17 (Th17) subset of lymphocytes has been implicated in a number of diseases that affect the nervous system. As the canonical cytokine of Th17 cells, interleukin 17A (IL-17A) is thought to contribute to these neuroimmune interactions. The main receptor for IL-17A is expressed in many neural tissues. IL-17A has direct effects on neurons but can also impact neural function via signaling to satellite cells and immune cells. In the central nervous system, IL-17A has been associated with neuropathology in multiple sclerosis, epilepsy syndromes and ischemic brain injury. Effects of IL-17A at the level of dorsal root ganglia and the spinal cord may contribute to enhanced nociception during neuropathic and inflammatory pain. Finally, IL-17A plays a role in sympathetic axon growth and regeneration of damaged axons that innervate the cornea. Given the widespread effects of IL-17A on neural tissues, it will be important to determine whether selectively mitigating the damaging effects of this cytokine while augmenting its beneficial effects is a possible strategy to treat inflammatory damage to the nervous system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.

The sympathetic innervation of the gastrointestinal (GI) tract regulates motility, secretion and blood flow by inhibiting the activity of the enteric nervous system (ENS) and direct vasoconstrictor innervation of the gut microvasculature. In addition to these well-established roles, there is evidence that the sympathetic nervous system (SNS) can modulate GI inflammation. Postganglionic sympathetic neurons innervate lymphoid tissues and immune cells within the GI tract. Furthermore, innate and adaptive immune cells express receptors for sympathetic neurotransmitters. Activation of these receptors can affect a variety of important immune cell functions, including cytokine release and differentiation of helper T lymphocyte subsets. This review will consider the neuroanatomical evidence of GI immune cell innervation by sympathetic axons, the effects of blocking or enhancing SNS activity on GI inflammation, and the converse modulation of sympathetic neuroanatomy and function by GI inflammation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Enhanced epinephrine secretion from adrenal chromaffin cells (ACCs) is an important homeostatic response to severe systemic inflammation during sepsis. Evidence suggests that increased activation of ACCs by preganglionic sympathetic neurons and direct alterations in ACC function contribute to this response. However, the direct effects of sepsis on ACC function have yet to be characterized. We hypothesized that sepsis enhances epinephrine secretion from ACCs by increasing intracellular Ca(2+) signaling. Plasma epinephrine concentration was increased 5-fold in the lipopolysaccharide-induced endotoxemia model of sepsis compared with saline-treated control mice. Endotoxemia significantly enhanced stimulus-evoked epinephrine secretion from isolated ACCs in vitro. Carbon fiber amperometry revealed an increase in the number of secretory events during endotoxemia, without significant changes in spike amplitude, half-width, or quantal content. ACCs isolated up to 12 hours after the induction of endotoxemia exhibited larger stimulus-evoked Ca(2+) transients compared with controls. Similarly, ACCs from cecal ligation and puncture mice also exhibited enhanced Ca(2+) signaling. Although sepsis did not significantly affect ACC excitability or voltage-gated Ca(2+) currents, a 2-fold increase in caffeine (10 mM)-stimulated Ca(2+) transients was observed during endotoxemia. Depletion of endoplasmic reticulum Ca(2+) stores using cyclopiazonic acid (10 μM) abolished the effects of endotoxemia on catecholamine secretion from ACCs. These findings suggest that sepsis directly enhances catecholamine secretion from ACCs through an increase in Ca(2+) release from the endoplasmic reticulum. These alterations in ACC function are likely to amplify the effects of increased preganglionic sympathetic neuron activity to further enhance epinephrine levels during sepsis.

Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease lead to altered gastrointestinal (GI) function as a consequence of the effects of inflammation on the tissues that comprise the GI tract. Among these tissues are several types of neurons that detect the state of the GI tract, transmit pain, and regulate functions such as motility, secretion, and blood flow. This review article describes the structure and function of the enteric nervous system, which is embedded within the gut wall, the sympathetic motor innervation of the colon and the extrinsic afferent innervation of the colon, and considers the evidence that colitis alters these important sensory and motor systems. These alterations may contribute to the pain and altered bowel habits that accompany IBD.

The enteric nervous system (ENS) is a vital part of the autonomic nervous system that regulates many gastrointestinal functions, including motility and secretion. All neurons and glia of the ENS arise from neural crest-derived cells that migrate into the gastrointestinal tract during embryonic development. It has been known for many years that a subpopulation of the enteric neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development. Recent studies have demonstrated that some enteric neurons exhibit electrical activity from as early as E11.5 in the mouse, with further maturation of activity during embryonic and postnatal development. This article discusses the maturation of electrophysiological and morphological properties of enteric neurons, the formation of synapses and synaptic activity, and the influence of neural activity on ENS development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Endogenous opioids are implicated in pain-regulation in chronic inflammatory bowel disease (IBD). We sought to examine whether endogenous opioids suppress the excitability of colonic nociceptive dorsal root ganglia (DRG) neurons during chronic IBD, and if so, whether modulation of underlying voltage-gated K(+) currents was involved., Methods: The effects of chronic dextran sulfate sodium (DSS) colitis on afferent signaling in mice was studied using patch clamp recordings. Colonic DRG neurons were identified using Fast Blue retrograde labeling and recordings obtained from small DRG neurons (<40 pF)., Key Results: In current-clamp recordings, the rheobase of neurons was increased 47% (P < 0.01) and action potential discharge at twice rheobase decreased 23% (P < 0.05) following incubation in colonic supernatants from chronic DSS mice. β-endorphin increased 14-fold, and tissue opioid immunoreactivity and expression in CD4+ cells observed by flow cytometry increased in chronic DSS colons. Incubation of naïve neurons in the μ-opioid receptor agonist D-Ala(2), N- MePhe(4), Gly-ol (DAMGO) (10 nM) partially recapitulated the effects of supernatants from DSS mice on rheobase. Supernatant effects were blocked by the μ-opioid receptor antagonist naloxone. In voltage clamp, chronic DSS supernatants and DAMGO increased I(A) K(+) currents., Conclusions & Inferences: The release of endogenous opioids during chronic inflammation in mice suppresses the excitability of nociceptive DRG neurons. Targeting immune cells may provide a novel means of modulating IBD pain., (© 2012 Blackwell Publishing Ltd.)

The adrenal medulla contains fenestrated capillaries that allow catecholamines and neuropeptides secreted by adrenal chromaffin cells (ACCs) to readily access the circulation. These capillaries may also allow bacterial products to enter the adrenal medulla and interact with ACCs during infection. One potential mediator of this interaction is toll-like receptor 4 (TLR-4), a pattern-recognition receptor that detects lipopolysaccharide (LPS) from Gram-negative bacteria. Evidence suggests that excitable cells can express TLR-4 and that LPS can modulate important neuronal and endocrine functions. The present study was therefore performed to test the hypothesis that TLR-4 activation by LPS affects ACC excitability and secretory output. RT-PCR revealed that TLR-4, cluster of differentiation 14, myeloid differentiation protein-2, and myeloid-derived factor 88 are expressed within mouse adrenal medullae. TLR-4 immunoreactivity was observed within all tyrosine hydroxylase immunoreactive ACCs. Incubation of isolated ACCs in LPS dose dependently hyperpolarized the resting membrane potential and enhanced large conductance (BK) Ca(2+)-activated K(+) currents. LPS (10 μg/ml) also increased rheobase, decreased the number of action potentials fired at rheobase, and reduced the percentage of ACCs exhibiting spontaneous and anodal break action potentials. Although catecholamine release was unaltered, LPS significantly reduced high-K(+)-stimulated neuropeptide Y release from isolated ACCs. LPS did not alter the excitability of ACCs from TLR-4(-/-) mice. Inhibition of nuclear factor-κB signaling with SC-514 (20 μm) abolished the effects of LPS on ACC excitability. Our findings suggest that LPS acts at TLR-4 to reduce ACC excitability and neuropeptide Y release through an nuclear factor-κB-dependent pathway.

Extracellular purines play important roles as neurotransmitters and paracrine mediators in the gastrointestinal (GI) tract. Inflammation of the GI tract causes marked changes in the release and extracellular catabolism of purines, and can modulate purinoceptor expression and/or signaling. The functional consequences of this include suppression of the purinergic component of inhibitory neuromuscular and neurovascular transmission, increased release of purines from immune and epithelial cells, loss of enteric neurons to damage through P2X(7) purinoceptors, and enhanced activation of pain fibres. The purinergic system represents an important target for drug therapies that may improve GI inflammation and its consequences., (Copyright © 2012. Published by Elsevier Ltd.)

Neural activity is integral to the development of the enteric nervous system (ENS). A subpopulation of neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development (at E10.5 in the mouse). However, the electrical activity of these cells has not been previously characterized, and it is not known whether all cells expressing neuronal markers are capable of firing action potentials (APs). In this study, we examined the activity of "neuron"-like cells (expressing pan-neuronal markers or with neuronal morphology) in the gut of E11.5 and E12.5 mice using whole-cell patch-clamp electrophysiology and compared them to the activity of neonatal and adult enteric neurons. Around 30-40% of neuron-like cells at E11.5 and E12.5 fired APs, some of which were very similar to those of adult enteric neurons. All APs were sensitive to tetrodotoxin (TTX), indicating that they were driven by voltage-gated Na+ currents. Expression of mRNA encoding several voltage-gated Na+ channels by the E11.5 gut was detected using RT-PCR. The density of voltage-gated Na+ currents increased from E11.5 to neonates. Immature active responses, mediated in part by TTX- and lidocaine-insensitive channels, were observed in most cells at E11.5 and E12.5, but not in P0/P1 or adult neurons. However, some cells expressing neuronal markers at E11.5 or E12.5 did not exhibit an active response to depolarization. Spontaneous depolarizations resembling excitatory postsynaptic potentials were observed at E12.5. The ENS is one of the earliest parts of the developing nervous system to exhibit mature forms of electrical activity.

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.

Inflammation can profoundly alter the structure and function of the nervous system. Interleukin (IL)-17 has been implicated in the pathogenesis of several inflammatory diseases associated with nervous system plasticity. However, the effects of IL-17 on the nervous system remain unexplored. Cell and explant culture techniques, immunohistochemistry, electrophysiology, and Ca2+ imaging were used to examine the impact of IL-17 on adult mouse sympathetic neurons. Receptors for IL-17 were present on postganglionic neurons from superior mesenteric ganglia (SMG). Supernatant from activated splenic T lymphocytes, which was abundant in IL-17, dramatically enhanced axonal length of SMG neurons. Importantly, IL-17-neutralizing antiserum abrogated the neurotrophic effect of splenocyte supernatant, and incubation of SMG neurons in IL-17 (1 ng/ml) significantly potentiated neurite outgrowth. The neurotrophic effect of IL-17 was accompanied by inhibition of voltage-dependent Ca2+ influx and was recapitulated by incubation of neurons in a blocker of N-type Ca2+ channels (ω-conotoxin GVIA; 30 nM). IL-17-induced neurite outgrowth in vitro appeared to be independent of glia, as treatment with a glial toxin (AraC; 5 μM) did not affect the outgrowth response to IL-17. Moreover, application of the cytokine to distal axons devoid of glial processes enhanced neurite extension. An inhibitor of the NF-κB pathway (SC-514; 20 μM) blocked the effects of IL-17. These data represent the first evidence that IL-17 can act on sympathetic somata and distal neurites to enhance neurite outgrowth, and identify a novel potential role for IL-17 in the neuroanatomical plasticity that accompanies inflammation.

The sympathetic nervous system regulates visceral function through the release of catecholamines and cotransmitters from postganglionic sympathetic neurons and adrenal chromaffin cells (ACCs). Previous studies have shown that norepinephrine secretion is decreased during experimental colitis due to the inhibition of voltage-gated Ca(2+) current (I(Ca)) in postganglionic sympathetic neurons. The present study examined whether colonic inflammation causes a similar impairment in depolarization-induced Ca(2+) influx in ACCs using the dextran sulfate sodium (DSS) model of acute colitis in mice. Alterations in ACC function during colitis were assessed using fura 2-acetoxymethyl ester Ca(2+) imaging techniques and perforated patch-clamp electrophysiology. In ACCs isolated from mice with DSS-induced acute colitis, the high-K(+)-stimulated increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was significantly reduced to 74% of the response of ACCs from control mice. Acute colitis caused a 10-mV hyperpolarization of ACC resting membrane potential, without a significant effect on cellular excitability. Delayed-rectifier K(+) and voltage-gated Na(+) current densities were significantly enhanced in ACCs from mice with DSS-induced acute colitis, with peak current densities of 154 and 144% that of controls, respectively. Importantly, acute colitis significantly inhibited I(Ca) in ACCs between -25 and +20 mV. Peak I(Ca) density in ACCs from mice with DSS-induced acute colitis was 61% that of controls. High-K(+)-induced increases in [Ca(2+)](i) were also reduced in ACCs from mice with 2,4,6-trinitrobenzene sulfonic acid-induced acute colitis and DSS-induced chronic colitis to 68 and 78% of the control responses, respectively. Our results suggest that, during colitis, voltage-dependent Ca(2+) influx is impaired in ACCs. Given the importance of Ca(2+) signaling in exocytosis, these alterations may decrease systemic catecholamine levels, which could play an important role in inflammatory bowel disease. This is the first demonstration of aberrant ACC function during experimental colitis.