Activation of MrgprA3 and MrgprC11 on Bladder-Innervating Afferents Induces Peripheral and Central Hypersensitivity to Bladder Distension.

Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterΔ-/- mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterΔ-/- mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development.SIGNIFICANCE STATEMENT Determining how bladder afferents become sensitized is the first step in finding effective treatments for common urological disorders such as overactive bladder and interstitial cystitis/bladder pain syndrome. Here we show that two of the key receptors, MrgprA3 and MrgprC11, that mediate itch from the skin are also expressed on afferents innervating the bladder. Activation of these receptors results in sensitization of bladder afferents, resulting in sensory signals being sent into the spinal cord that prematurely indicate bladder fullness. Targeting bladder afferents expressing MrgprA3 or MrgprC11 and preventing their sensitization may provide a novel approach for treating overactive bladder and interstitial cystitis/bladder pain syndrome.

Trends in characteristics of fentanyl-related poisonings in the United States, 2015-2021.

Background: Fentanyl-related deaths continue to increase in the United States; however, most national studies focus on fatal overdose. More research, including data on nonfatal overdose, is needed.Objective: We examined trends in characteristics of fatal and nonfatal fentanyl-related poisonings ("exposures") in the US.Methods: National Poison Control data were examined to estimate trends in characteristics of reported exposures between 2015 and 2021 (N = 15,391; 38.7% female). We also delineated correlates of experiencing a major adverse effect or death.Results: The proportion of exposures increased among all age groups between ages 13 and 39 (ps < .05) with the largest increase among those age 13-19 (a 127.8% increase). With respect to reasons for use, the proportion of cases involving fentanyl "abuse" increased by 63.8% (p < .001). The proportion involving fentanyl inhalation increased 427.6% from 5.7% to 29.9% and injection increased from 6.7% to 9.6%, a 42.3% increase (ps < .01). The proportion also increased for co-use of methamphetamine (by 669.0%), cocaine (by 374.0%), and heroin (by 159.5%). The proportion of major adverse effects increased from 15.5% to 39.6% (p < .001). In the multivariable model, "abuse", suspected suicide attempts, and use via inhalation were risk factors for experiencing a major effect or death, and misuse, ingestion, dermal use, and co-use of methamphetamine were associated with lower risk.Conclusion: Poison Control data suggest that characteristics of individuals exposed to fentanyl continue to shift, with use via inhalation increasing and medical outcomes of nonfatal poisonings becoming more severe. These results complement mortality data and inform prevention and harm reduction efforts.

Deoxycholic acid activates colonic afferent nerves via 5-HT3 receptor-dependent and -independent mechanisms.

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-HT3 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.

NKA enhances bladder-afferent mechanosensitivity via urothelial and detrusor activation.

Tachykinins are expressed within bladder-innervating sensory afferents and have been shown to generate detrusor contraction and trigger micturition. The release of tachykinins from these sensory afferents may also activate tachykinin receptors on the urothelium or sensory afferents directly. Here, we investigated the direct and indirect influence of tachykinins on mechanosensation by recording sensory signaling from the bladder during distension, urothelial transmitter release ex vivo, and direct responses to neurokinin A (NKA) on isolated mouse urothelial cells and bladder-innervating DRG neurons. Bath application of NKA induced concentration-dependent increases in bladder-afferent firing and intravesical pressure that were attenuated by nifedipine and by the NK2 receptor antagonist GR159897 (100 nM). Intravesical NKA significantly decreased bladder compliance but had no direct effect on mechanosensitivity to bladder distension (30 µl/min). GR159897 alone enhanced bladder compliance but had no effect on mechanosensation. Intravesical NKA enhanced both the amplitude and frequency of bladder micromotions during distension, which induced significant transient increases in afferent firing, and were abolished by GR159897. NKA increased intracellular calcium levels in primary urothelial cells but not bladder-innervating DRG neurons. Urothelial ATP release during bladder distention was unchanged in the presence of NKA, whereas acetylcholine levels were reduced. NKA-mediated activation of urothelial cells and enhancement of bladder micromotions are novel mechanisms for NK2 receptor-mediated modulation of bladder mechanosensation. These results suggest that NKA influences bladder afferent activity indirectly via changes in detrusor contraction and urothelial mediator release. Direct actions on sensory nerves are unlikely to contribute to the effects of NKA.

Fundamentals of Neurogastroenterology: Basic Science.

This review examines the fundamentals of neurogastroenterology that may underlie the pathophysiology of functional GI disorders (FGIDs). It was prepared by an invited committee of international experts and represents an abbreviated version of their consensus document that will be published in its entirety in the forthcoming book and online version entitled ROME IV. It emphasizes recent advances in our understanding of the enteric nervous system, sensory physiology underlying pain, and stress signaling pathways. There is also a focus on neuroimmmune signaling and intestinal barrier function, given the recent evidence implicating the microbiome, diet, and mucosal immune activation in FGIDs. Together, these advances provide a host of exciting new targets to identify and treat FGIDs and new areas for future research into their pathophysiology.

Gastrointestinal Physiology and Function.

The gastrointestinal (GI) system is responsible for the digestion and absorption of ingested food and liquids. Due to the complexity of the GI tract and the substantial volume of material that could be covered under the scope of GI physiology, this chapter briefly reviews the overall function of the GI tract, and discusses the major factors affecting GI physiology and function, including the intestinal microbiota, chronic stress, inflammation, and aging with a focus on the neural regulation of the GI tract and an emphasis on basic brain-gut interactions that serve to modulate the GI tract. GI diseases refer to diseases of the esophagus, stomach, small intestine, colon, and rectum. The major symptoms of common GI disorders include recurrent abdominal pain and bloating, heartburn, indigestion/dyspepsia, nausea and vomiting, diarrhea, and constipation. GI disorders rank among the most prevalent disorders, with the most common including esophageal and swallowing disorders, gastric and peptic ulcer disease, gastroparesis or delayed gastric emptying, irritable bowel syndrome (IBS), and inflammatory bowel disease (IBD). Many GI disorders are difficult to diagnose and their symptoms are not effectively managed. Thus, basic research is required to drive the development of novel therapeutics which are urgently needed. One approach is to enhance our understanding of gut physiology and pathophysiology especially as it relates to gut-brain communications since they have clinical relevance to a number of GI complaints and represent a therapeutic target for the treatment of conditions including inflammatory diseases of the GI tract such as IBD and functional gut disorders such as IBS.

Ageing and gastrointestinal sensory function: altered colonic mechanosensory and chemosensory function in the aged mouse.

KEY POINTS: Remarkably little is known about how age affects the sensory signalling pathways in the gastrointestinal tract despite age-related gastrointestinal dysfunction being a prime cause of morbidity amongst the elderly population High-threshold gastrointestinal sensory nerves play a key role in signalling distressing information from the gut to the brain. We found that ageing is associated with attenuated high-threshold afferent mechanosensitivity in the murine colon, and associated loss of TRPV1 channel function. These units have the capacity to sensitise in response to injurious events, and their loss in ageing may predispose the elderly to lower awareness of GI injury or disease. ABSTRACT: Ageing has a profound effect upon gastrointestinal function through mechanisms that are poorly understood. Here we investigated the effect of age upon gastrointestinal sensory signalling pathways in order to address the mechanisms underlying these changes. In vitro mouse colonic and jejunal preparations with attached splanchnic and mesenteric nerves were used to study mechanosensory and chemosensory afferent function in 3-, 12- and 24-month-old C57BL/6 animals. Quantitative RT-PCR was used to investigate mRNA expression in colonic tissue and dorsal root ganglion (DRG) cells isolated from 3- and 24-month animals, and immunohistochemistry was used to quantify the number of 5-HT-expressing enterochromaffin (EC) cells. Colonic and jejunal afferent mechanosensory function was attenuated with age and these effects appeared earlier in the colon compared to the jejunum. Colonic age-related loss of mechanosensory function was more pronounced in high-threshold afferents compared to low-threshold afferents. Chemosensory function was attenuated in the 24-month colon, affecting TRPV1 and serotonergic signalling pathways. High-threshold mechanosensory afferent fibres and small-diameter DRG neurons possessed lower functional TRPV1 receptor responses, which occurred without a change in TRPV1 mRNA expression. Serotonergic signalling was attenuated at 24 months, but TPH1 and TPH2 mRNA expression was elevated in colonic tissue. In conclusion, we saw an age-associated decrease in afferent mechanosensitivity in the mouse colon affecting HT units. These units have the capacity to sensitise in response to injurious events, and their loss in ageing may predispose the elderly to lower awareness of GI injury or disease.

Interdependency between mechanical parameters and afferent nerve discharge in hypertrophic intestine of rats.

Partial intestinal obstruction causes smooth muscle hypertrophy, enteric neuronal plasticity, motility disorders, and biomechanical remodeling. In this study we characterized the stimulus-response function of afferent fibers innervating the partially obstructed jejunum. A key question is whether changes in afferent firing arise from remodeled mechanical tissue properties or from adaptive afferent processes. Partial obstruction was created by placing a polyethylene ring for 2 wk in jejunum of seven rats. Sham obstruction was made in six rats and seven rats served as normal controls. Firing from mesenteric afferent nerve bundles was recorded during mechanical ramp, relaxation, and creep tests. Stress-strain, spike rate increase ratio (SRIR), and firing rate in single units were assessed for evaluation of interdependency of the mechanical stimulations, histomorphometry data, and afferent nerve discharge. Partial intestinal obstruction resulted in hypertrophy and jejunal stiffening proximal to the obstruction site. Low SRIR at low strains during fast distension and at high stresses during slow distension was found in the obstructed rats. Single unit analysis showed increased proportion of mechanosensitive units but absent high-threshold (HT) units during slow stimulation, decreased number of HT units during fast stimulation, and shift from HT sensitivity towards low threshold sensitivity in the obstructed jejunum. Biomechanical remodeling and altered afferent response to mechanical stimulations were found in the obstructed jejunum. Afferents from obstructed jejunum preserved their function in encoding ongoing mechanical stimulation but showed changes in their responsiveness. The findings support that mechanical factors rather than adaption are important for afferent remodeling.

Ageing and Gastrointestinal Sensory Function.

Over the past few decades a combination of electrophysiological, morphological and molecular approaches has enabled the different populations of vagal and spinal afferents that innervate the bowel to be characterized. The sensitivity of these afferents is determined by their location in the gut wall, their relationship with other cells and structures and the receptors and ion channels that they express on their nerve terminals. An important feature of this innervation is that it is upregulated during injury, inflammation and ischaemia through changes in receptors and ion channels that determine excitability and sensitivity. In recent studies we have sought to identify how sensory mechanisms are influenced as part of the normal ageing process. Attenuated signaling was evident in different gastrointestinal afferent subpopulations conveying low and high threshold mechanosensory information and there was impairment in the ability of sensory neurons to sensitize in response to chemical mediators such as 5-HT. These sensory deficits may contribute to altered bowel habits with age and the prevalence of incontinence in the elderly.

Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology.

The Journal of Physiology and Experimental Physiology have always used UK legislation as the basis of their policy on ethical standards in experiments on non-human animals. However, for international journals with authors, editors and referees from outside the UK the policy can lack transparency and is sometimes cumbersome, requiring the intervention of a Senior Ethics Reviewer or advice from external experts familiar with UK legislation. The journals have therefore decided to set out detailed guidelines for how authors should report experimental procedures that involve animals. As well as helping authors, this new clarity will facilitate the review process and decision making where there are questions regarding animal ethics.

Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology.

The Journal of Physiology and Experimental Physiology have always used UK legislation as the basis of their policy on ethical standards in experiments on non-human animals. However, for international journals with authors, editors and referees from outside the UK the policy can lack transparency and is sometimes cumbersome, requiring the intervention of a Senior Ethics Reviewer or advice from external experts familiar with UK legislation. The journals have therefore decided to set out detailed guidelines for how authors should report experimental procedures that involve animals. As well as helping authors, this new clarity will facilitate the review process and decision making where there are questions regarding animal ethics.

Age-related changes in afferent pathways and urothelial function in the male mouse bladder.

The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea.

Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity.

Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo- and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca(2+) dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both types of neurons). In addition, we analysed the association between neuronal activation and individual discomfort/pain pressure thresholds. The IBS supernatants evoked Ca(2+) transients in DRG neurons and spike discharge in submucous neurons. Submucous and DRG neurons showed significantly stronger responses to supernatants from hypersensitive IBS patients as reflected by higher spike frequency or stronger [Ca(2+)]i transients in a larger proportion of neurons. The neuroindex as a product of spike frequency or [Ca(2+)]i transients and proportion of responding neurons correlated significantly with the individual discomfort/pain thresholds of the IBS patients. Supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons. The level of activation correlated with the individual discomfort/pain threshold pressure values. These findings support our hypothesis that visceral sensitivity is linked to activation of peripheral neurons by biopsy supernatants.

Evidence for stress-dependent mechanoreceptors linking intestinal biomechanics and sensory signal transduction.

Sensory nerve endings are widely distributed throughout the body. Neither the nature of the mechanosensitive channels nor the principal mechanical stimulus for these receptors is known. Afferents supplying the gastrointestinal tract responding to distension and contraction are responsible for co-ordinated reflex control, feeding behaviour and sensations, including pain. Different populations of intestinal afferent fibres follow different pathways to the CNS, have different terminal fields and possess different thresholds for activation that may reflect the extent to which mechanical forces are distributed and dissipated by non-neural structures in the bowel wall. In this study, we have characterized the stimulus-response function of afferent fibres innervating the rat jejunum, correlating luminal distensions in the bowel wall with the firing frequency of mesenteric afferent nerve bundles. Combining video imaging with intraluminal pressure recordings and utilizing a strain softening protocol, we have determined whether mechanoreceptors respond primarily to stress or strain. Multiunit afferent recordings were separated using spike discrimination software into low-threshold (LT) and high-threshold (HT) single units. For multifibre afferent recordings and both LT and HT single units, we observed a linear relationship between circumferential stress and mesenteric afferent discharge that was independent of distension-induced tissue softening, with correlation coefficients >0.9. A fivefold change in the rate of applied distension did not significantly alter the magnitude of the afferent response and the linearity of the stress-dependent mechanotransduction in both multifibre preparations and the LT and HT afferent fibres (P > 0.2). Thus, the firing characteristics of intestinal mechanoreceptors are linearly associated with the input in terms of mechanical stress.

OnabotulinumtoxinA significantly attenuates bladder afferent nerve firing and inhibits ATP release from the urothelium.

OBJECTIVE: To investigate the direct effect of onabotulinumtoxinA (OnaBotA) on bladder afferent nerve activity and release of ATP and acetylcholine (ACh) from the urothelium. MATERIALS AND METHODS: Bladder afferent nerve activity was recorded using an in vitro mouse preparation enabling simultaneous recordings of afferent nerve firing and intravesical pressure during bladder distension. Intraluminal and extraluminal ATP, ACh, and nitric oxide (NO) release were measured using the luciferin-luciferase and Amplex(®) Red assays (Molecular Probes, Carlsbad, CA, USA), and fluorometric assay kit, respectively. OnaBotA (2U), was applied intraluminally, during bladder distension, and its effect was monitored for 2 h after application. Whole-nerve activity was analysed to classify the single afferent units responding to physiological (low-threshold [LT] afferent <15 mmHg) and supra-physiological (high-threshold [HT] afferent >15 mmHg) distension pressures. RESULTS: Bladder distension evoked reproducible pressure-dependent increases in afferent nerve firing. After exposure to OnaBotA, both LT and HT afferent units were significantly attenuated. OnaBotA also significantly inhibited ATP release from the urothelium and increased NO release. CONCLUSION: These data indicate that OnaBotA attenuates the bladder afferent nerves involved in micturition and bladder sensation, suggesting that OnaBotA may exert its clinical effects on urinary urgency and the other symptoms of overactive bladder syndrome through its marked effect on afferent nerves.

P2X7 receptor-dependent intestinal afferent hypersensitivity in a mouse model of postinfectious irritable bowel syndrome.

The ATP-gated P2X(7) receptor (P2X(7)R) was shown to be an important mediator of inflammation and inflammatory pain through its regulation of IL-1ß processing and release. Trichinella spiralis-infected mice develop a postinflammatory visceral hypersensitivity that is reminiscent of the clinical features associated with postinfectious irritable bowel syndrome. In this study, we used P2X(7)R knockout mice (P2X(7)R(-/-)) to investigate the role of P2X(7)R activation in the in vivo production of IL-1ß and the development of postinflammatory visceral hypersensitivity in the T. spiralis-infected mouse. During acute nematode infection, IL-1ß-containing cells and P2X(7)R expression were increased in the jejunum of wild-type (WT) mice. Peritoneal and serum IL-1ß levels were also increased, which was indicative of elevated IL-1ß release. However, in the P2X(7)R(-/-) animals, we found that infection had no effect upon intracellular, plasma, or peritoneal IL-1ß levels. Conversely, infection augmented peritoneal TNF-α levels in both WT and P2X(7)R(-/-) animals. Infection was also associated with a P2X(7)R-dependent increase in extracellular peritoneal lactate dehydrogenase, and it triggered immunological changes in both strains. Jejunal afferent fiber mechanosensitivity was assessed in uninfected and postinfected WT and P2X(7)R(-/-) animals. Postinfected WT animals developed an augmented afferent fiber response to mechanical stimuli; however, this did not develop in postinfected P2X(7)R(-/-) animals. Therefore, our results demonstrated that P2X(7)Rs play a pivotal role in intestinal inflammation and are a trigger for the development of visceral hypersensitivity.

Characteristics of poisonings involving ketamine in the United States, 2019-2021.

BACKGROUND: The use of ketamine, a controlled dissociative anesthetic, has become more widespread in recent years with recreational/nonmedical use increasing and ketamine becoming more widely available in clinics to treat depression. AIMS: We examined recent trends in adverse effects related to ketamine use. METHODS: US National Poison Control data were examined, focusing on ketamine exposures among those aged ⩾13 between 2019 and 2021 (n = 758). We examined quarterly trends in exposure and delineated correlates of patients experiencing a major adverse effect or death. RESULTS: The number of reported exposures increased 81.1% from 2019 Quarter 1 through 2021 Quarter 4, from 37 to 67 (p = 0.018). The majority of patients were male (57.1%), and the plurality of cases involved intentional misuse or "abuse" (39.5%), followed by suspected suicide attempt (19.7%) and unintentional exposure (18.9%). A fifth (19.6%) experienced a major adverse effect or death. A third (33.4%) co-used other drugs; the drugs most commonly co-used were benzodiazepines (14.6%), alcohol (10.3%), and opioids (8.7%). Co-use of gamma-hydroxybutyrate (GHB; adjusted prevalence ratio (aPR) = 3.43, 95% confidence interval (CI): 1.57-7.46) and opioids (aPR = 2.44, 95% CI: 1.46-4.08) was associated with increased risk for a major adverse effect or death, as was injection-only administration (aPR = 2.68, 95% CI: 1.21-5.92). CONCLUSIONS: Although still rare, poisonings involving ketamine have increased in recent years. Polydrug use-particularly with opioids or GHB-appears to be a particular risk factor for more serious adverse effects. As prevalence of use increases, it is important to monitor adverse effects and co-occurring behaviors to inform timely prevention and harm reduction as needed.

Mast cells drive mesenteric afferent signalling during acute intestinal ischaemia.

Acute intestinal ischaemia stimulates visceral afferent nerves but the mechanisms responsible for this excitation are not fully understood. Mast cells may participate in this process as they are known to signal to mesenteric afferents during intestinal anaphylaxis and contribute to early inflammation and neuronal damage in response to cerebral ischaemia. We therefore hypothesised that mast cells are early responders to acute intestinal ischaemia and their activation initiates rapid signalling to the CNS via the excitation of mesenteric afferents. Primary afferent firing was recorded from a mesenteric nerve bundle supplying a segment of jejunum in anaesthetized adult rats. Acute focal ischaemia was produced by clamping theme senteric vessels for 8 min, and reperfusion followed removal of the vessel clip. Two episodes of ischaemia­reperfusion (I­R) separated by a 30 min interval were performed. Drugs or their vehicles were administered 10 min before the 2nd I­R episode. Ischaemia caused a reproducible, intense and biphasic afferent firing that was temporally dissociated from the concomitantly triggered complex pattern of intestinal motor activity. The L-type calcium channel blocker, nifedipine, significantly attenuated this afferent firing by a mechanism independent of its action on intestinal tone. Ischaemia-induced afferent firing was also abrogated by the mast cell stabilizer, doxantrazole, and the H1 histamine receptor antagonist, pyrilamine. In contrast, the nicotinic receptor antagonist, hexamethonium, and the N-type calcium channel toxin, ω-conotoxin GVIA, each reduced the ischaemia-evoked motor inhibition but not the concurrent afferent discharge. Similarly, the cyclooxygenase inhibitor, naproxen, had no effect on the ischaemic afferent response but reduced the intestinal tone shortly from the onset of ischaemia to the early period of reperfusion. These data support a critical role for mast cell-derived histamine in the direct chemoexcitation of mesenteric afferents during acute intestinal ischaemia, whereas enteric reflex mechanisms and cyclooxygenase products contribute primarily to ischaemia-induced changes in intestinal motility. Therefore, targeting mast cells may provide benefits in patients with abdominal pain resulting from an ischaemic insult to the gastrointestinal tract.

Intestinal serotonin release, sensory neuron activation, and abdominal pain in irritable bowel syndrome.

OBJECTIVES: Serotonin (5-hydroxytryptamine, 5-HT) metabolism may be altered in gut disorders, including in the irritable bowel syndrome (IBS). We assessed in patients with IBS vs. healthy controls (HCs) the number of colonic 5-HT-positive cells; the amount of mucosal 5-HT release; their correlation with mast cell counts and mediator release, as well as IBS symptoms; and the effects of mucosal 5-HT on electrophysiological responses in vitro. METHODS: We enrolled 25 Rome II IBS patients and 12 HCs. IBS symptom severity and frequency were graded 0-4. 5-HT-positive enterochromaffin cells and tryptase-positive mast cells were assessed with quantitative immunohistochemistry on colonic biopsies. Mucosal 5-HT and mast cell mediators were assessed by high-performance liquid chromatography or immunoenzymatic assay, respectively. The impact of mucosal 5-HT on electrophysiological activity of rat mesenteric afferent nerves was evaluated in vitro. RESULTS: Compared with HCs, patients with IBS showed a significant increase in 5-HT-positive cell counts (0.37 ± 0.16% vs. 0.56 ± 0.26%; P=0.039), which was significantly greater in patients with diarrhea-predominant IBS vs. constipation-predominant IBS (P=0.035). Compared with HCs, 5-HT release in patients with IBS was 10-fold significantly increased (P < 0.001), irrespective of bowel habit, and was correlated with mast cell counts. A significant correlation was found between the mucosal 5-HT release and the severity of abdominal pain (r(s)=0.582, P=0.047). The area under the curve, but not peak sensory afferent discharge evoked by IBS samples in rat jejunum, was significantly inhibited by the 5-HT3 receptor antagonist granisetron (P<0.005). CONCLUSIONS: In patients with IBS, 5-HT spontaneous release was significantly increased irrespective of bowel habit and correlated with mast cell counts and the severity of abdominal pain. Our results suggest that increased 5-HT release contributes to development of abdominal pain in IBS, probably through mucosal immune activation.