Regional differences in the ultrastructure of mucosal macrophages in the rat large intestine.

We previously clarified the histological characteristics of macrophages in the rat small intestine using serial block-face scanning electron microscopy (SBF-SEM). However, the regional differences in the characteristics of macrophages throughout the large intestine remain unknown. Here, we performed a pilot study to explore the regional differences in the ultrastructure of mucosal macrophages in the large intestine by using SBF-SEM analysis. SBF-SEM analysis conducted on the luminal side of the cecum and descending colon revealed macrophages as amorphous cells possessing abundant lysosomes and vacuoles. Macrophages in the cecum exhibited a higher abundance of lysosomes and a lower abundance of vacuoles than those in the descending colon. Macrophages with many intraepithelial cellular processes were observed beneath the intestinal superficial epithelium in the descending colon. Moreover, macrophages in contact with nerve fibers were more prevalent in the cecum than in the descending colon, and a subset of them surrounded a nerve bundle only in the cecum. In conclusion, the present pilot study suggested that the quantity of some organelles (lysosomes and vacuoles) in macrophages differed between the cecum and the descending colon and that there were some region-specific subsets of macrophages like nerve-associated macrophages in the cecum.

Disparities in the gut metabolome of post-operative Hirschsprung's disease patients.

Hirschsprung's disease (HD) is a congenital structural abnormality of the colon seen in approximately 1 to 5000 live births. Despite surgical correction shortly after presentation, up to 60% of patients will express long-term gastrointestinal complaints, including potentially life-threatening Hirschsprung-associated enterocolitis (HAEC). In this study fecal samples from postoperative HD patients (n = 38) and their healthy siblings (n = 21) were analysed using high-resolution liquid chromatography-mass spectrometry aiming to further unravel the nature of the chronic gastrointestinal disturbances. Furthermore, within the patient group, we compared the faecal metabolome between patients with and without a history of HAEC as well as those diagnosed with short or long aganglionic segment. Targeted analysis identified several individual metabolites characteristic for all HD patients as well as those with a history of HAEC and long segment HD. Moreover, multivariate models based on untargeted data established statistically significant (p < 0.05) differences in comprehensive faecal metabolome in the patients' cohort as a whole and in patients with a history of HAEC. Pathway analysis revealed the most impact on amino sugar, lysine, sialic acid, hyaluronan and heparan sulphate metabolism in HD, as well as impaired tyrosine metabolism in HAEC group. Those changes imply disruption of intestinal mucosal barrier due to glycosaminoglycan breakdown and dysbiosis as major metabolic changes in patients' group and should be further explored for potential diagnostic or treatment targets.

Understanding the physiology of human defaecation and disorders of continence and evacuation.

The act of defaecation, although a ubiquitous human experience, requires the coordinated actions of the anorectum and colon, pelvic floor musculature, and the enteric, peripheral and central nervous systems. Defaecation is best appreciated through the description of four phases, which are, temporally and physiologically, reasonably discrete. However, given the complexity of this process, it is unsurprising that disorders of defaecation are both common and problematic; almost everyone will experience constipation at some time in their life and many will develop faecal incontinence. A detailed understanding of the normal physiology of defaecation and continence is critical to inform management of disorders of defaecation. During the past decade, there have been major advances in the investigative tools used to assess colonic and anorectal function. This Review details the current understanding of defaecation and continence. This includes an overview of the relevant anatomy and physiology, a description of the four phases of defaecation, and factors influencing defaecation (demographics, stool frequency/consistency, psychobehavioural factors, posture, circadian rhythm, dietary intake and medications). A summary of the known pathophysiology of defaecation disorders including constipation, faecal incontinence and irritable bowel syndrome is also included, as well as considerations for further research in this field.

The Endocrine Disruptor Bisphenol A (BPA) Affects the Enteric Neurons Immunoreactive to Neuregulin 1 (NRG1) in the Enteric Nervous System of the Porcine Large Intestine.

The enteric nervous system (ENS), located in the wall of the gastrointestinal (GI) tract, is characterized by complex organization and a high degree of neurochemical diversity of neurons. One of the less known active neuronal substances found in the enteric neurons is neuregulin 1 (NRG1), a factor known to be involved in the assurance of normal development of the nervous system. During the study, made up using the double immunofluorescence technique, the presence of NRG1 in the ENS of the selected segment of porcine large intestine (caecum, ascending and descending colon) was observed in physiological conditions, as well as under the impact of low and high doses of bisphenol A (BPA) which is commonly used in the production of plastics. In control animals in all types of the enteric plexuses, the percentage of NRG1-positive neurons oscillated around 20% of all neurons. The administration of BPA caused an increase in the number of NRG1-positive neurons in all types of the enteric plexuses and in all segments of the large intestine studied. The most visible changes were noted in the inner submucous plexus of the ascending colon, where in animals treated with high doses of BPA, the percentage of NRG1-positive neurons amounted to above 45% of all neuronal cells. The mechanisms of observed changes are not entirely clear, but probably result from neurotoxic, neurodegenerative and/or proinflammatory activity of BPA and are protective and adaptive in nature.

The Neuropeptide System and Colorectal Cancer Liver Metastases: Mechanisms and Management.

Colorectal cancer (CRC), classified as the third most prevalent cancer worldwide, remains to be a clinical and research challenge. It is estimated that ~50% of CRC patients die from distant metastases, with treatment of this complication still posing significant difficulties. While liver metastasis (LM) cascade is known in the literature, its mechanisms are still unclear and remain studied in different research models. A connection is suggested between nervous system dysfunctions and a range of Neurotransmitters (Nts) (including Neuropeptides, NPs), Neurotrophins (Ntt) and their receptors (Rs) in CRC liver metastasis development. Studies on the role of NP/NP-Rs in the progression and metastasis of CRC, show the complexity of brain-tumor interactions, caused by their different forms of release to the extracellular environment (endocrine, autocrine, paracrine and neurocrine). Many stages of LM are connected to the activity of pro-inflammatory, e.g., Corticotropin-releasing Hormone Receptor 1 (CRHR1), Neuropeptide Y (NPY) and Neurotensin (NT), anti-inflammatory, e.g., Calcitonin Gene-related Peptide (CGRP), CRHR2 and Vasoactive Intestinal Polypeptide (VIP) or dual role neuropeptides, e.g., Substance P (SP). The regulation of the local immunological profile (e.g., CRH/CRHRs), dysfunctions of enteroprotective role of NPs on epithelial cells (e.g., NT/NT-R), as well as structural-functional changes in enteric nervous system innervation of the tumor are also important. More research is needed to understand the exact mechanisms of communication between the neurons and tumor cells. The knowledge on the mechanisms regulating tumor growth and different stages of metastasis, as well as effects of the action of a numerous group of Nts/NPs/Ntt as growth factors, have implications for future therapeutic strategies. To obtain the best treatment outcomes, it is important to use signaling pathways common for many NPs, as well to develop a range of broad-spectrum antagonists. This review aims to summarize the current knowledge on the importance of neuroactive molecules in the promotion of the invasion-metastasis cascade in CRC, as well as the improvements of clinical management of CRC liver metastasis.

Postnatal human enteric neuronal progenitors can migrate, differentiate, and proliferate in embryonic and postnatal aganglionic gut environments.

BACKGROUND: Enteric neural stem/progenitor cells (ENSCs) offer an innovative approach to treating Hirschsprung disease (HSCR) and other enteric neuropathies. However, postnatal-derived human ENSCs have not been thoroughly characterized and their behavior in the embryonic and postnatal intestinal environment is unknown. METHODS: ENSCs were isolated from the intestines of 25 patients undergoing bowel resection, including 7 children with HSCR. Neuronal differentiation and proliferation of ENSCs from submucosal and myenteric plexuses from patients with and without HSCR were characterized. ENSC migration and differentiation were studied following transplantation into embryonic chick neural crest, embryonic chick hindgut, and postnatal mouse aganglionic colon. RESULTS: The proliferative and neurogenic potential of ENSCs from HSCR intestine is equivalent to that of non-HSCR controls. Similarly, no difference was observed between myenteric- and submucosal-derived ENSCs. Postnatal ENSCs transplanted to embryonic neural crest pathways and to aneural hindgut migrate normally and differentiate into appropriate neural crest-derived cell types. ENSCs in postnatal mouse aganglionic colon differentiate into neurons and glia both ex vivo and in vivo. CONCLUSIONS: ENSCs isolated from the postnatal intestine of patients with and without HSCR can behave like embryonic neural crest-derived cells. These results support the feasibility of cell-based therapy for future treatment of neurointestinal disease.

Morphometric profile of large intestinal neuronal plexuses in normal perinatal autopsies and Hirschsprung disease.

OBJECTIVES: To derive objective values for the diagnosis of Hirschsprung disease (HSCR) from a comparison of the morphometric profile of large intestinal neuronal plexuses in normal perinatal autopsies and surgical specimens of HSCR. METHODS: A cross-sectional comparative study with 40 subjects each in (i) non-HSCR perinatal group encompassing neonates and stillborn babies beyond 30 weeks of gestation on whom autopsies were conducted and (ii) HSCR group comprising all patients clinicoradiologically diagnosed as HSCR. The morphometric assessment was done on hematoxylin-and-eosin-stained sections. KEY RESULTS: The morphometric profile in terms of average number of ganglia/linear mm of colon, interganglion distance, number of ganglion cells/ganglion, average ganglion cell length, ganglion cell nuclear area, ganglion cell nuclear diameter, nerve trunk thickness, and density has been outlined. On comparison with the neuroanatomically normal zone of HSCR, the cut-offs to identify hypertrophic nerve trunks (nerve trunk thickness of >37.85 µm) and reduced number of ganglia (number of ganglia/linear mm of colon <2.05 and interganglion distance of >229 µm) were derived. CONCLUSIONS & INFERENCES: The determined objective values, after testing on diagnostic rectal biopsies, may serve to formulate a diagnostic algorithm along with immunostaining for diagnosis of HSCR in colorectal specimens.

Identification of different functional types of spinal afferent neurons innervating the mouse large intestine using a novel CGRPα transgenic reporter mouse.

Spinal afferent neurons detect noxious and physiological stimuli in visceral organs. Five functional classes of afferent terminals have been extensively characterized in the colorectum, primarily from axonal recordings. Little is known about the corresponding somata of these classes of afferents, including their morphology, neurochemistry, and electrophysiology. To address this, we made intracellular recordings from somata in L6/S1 dorsal root ganglia and applied intraluminal colonic distensions. A transgenic calcitonin gene-related peptide-α (CGRPα)-mCherry reporter mouse, which enabled rapid identification of soma neurochemistry and morphology following electrophysiological recordings, was developed. Three distinct classes of low-threshold distension-sensitive colorectal afferent neurons were characterized; an additional group was distension-insensitive. Two of three low-threshold classes expressed CGRPα. One class expressing CGRPα discharged phasically, with inflections on the rising phase of their action potentials, at low frequencies, to both physiological (<30 mmHg) and noxious (>30 mmHg) distensions. The second class expressed CGRPα and discharged tonically, with smooth, briefer action potentials and significantly greater distension sensitivity than phasically firing neurons. A third class that lacked CGRPα generated the highest-frequency firing to distension and had smaller somata. Thus, CGRPα expression in colorectal afferents was associated with lower distension sensitivity and firing rates and larger somata, while colorectal afferents that generated the highest firing frequencies to distension had the smallest somata and lacked CGRPα. These data fill significant gaps in our understanding of the different classes of colorectal afferent somata that give rise to distinct functional classes of colorectal afferents. In healthy mice, the majority of sensory neurons that respond to colorectal distension are low-threshold, wide-dynamic-range afferents, encoding both physiological and noxious ranges.

Neural influences on human intestinal epithelium in vitro.

KEY POINTS: We present the first systematic and, up to now, most comprehensive evaluation of the basic features of epithelial functions, such as basal and nerve-evoked secretion, as well as tissue resistance, in over 2200 surgical specimens of human small and large intestine. We found no evidence for impaired nerve-evoked epithelial secretion or tissue resistance with age or disease pathologies (stomach, pancreas or colon cancer, polyps, diverticulitis, stoma reversal). This indicates the validity of future studies on epithelial secretion or resistance that are based on data from a variety of surgical specimens. ACh mainly mediated nerve-evoked and basal secretion in the small intestine, whereas vasoactive intestinal peptide and nitric oxide were the primary pro-secretory transmitters in the large intestine. The results of the present study revealed novel insights into regional differences in nerve-mediated secretion in the human intestine and comprise the basis by which to more specifically target impaired epithelial functions in the diseased gut. ABSTRACT: Knowledge on basic features of epithelial functions in the human intestine is scarce. We used Ussing chamber techniques to record basal tissue resistance (R-basal) and short circuit currents (ISC; secretion) under basal conditions (ISC-basal) and after electrical field stimulation (ISC-EFS) of nerves in 2221 resectates from 435 patients. ISC-EFS was TTX-sensitive and of comparable magnitude in the small and large intestine. ISC-EFS or R-basal were not influenced by the patients' age, sex or disease pathologies (cancer, polyps, diverticulitis). Ion substitution, bumetanide or adenylate cyclase inhibition studies suggested that ISC-EFS depended on epithelial cAMP-driven chloride and bicarbonate secretion but not on amiloride-sensitive sodium absorption. Although atropine-sensitive cholinergic components prevailed for ISC-EFS of the duodenum, jejunum and ileum, PG97-269-sensitive [vasoactive intestinal peptide (VIP) receptor 1 antagonist] VIPergic together with L-NAME-sensitive nitrergic components dominated the ISC-EFS in colonic preparations. Differences in numbers of cholinergic or VIPergic neurons, sensitivity of epithelial muscarinic or VIP receptors, or stimulus frequency-dependent transmitter release were not responsible for the region-specific transmitter contribution to ISC-EFS. Instead, the low atropine-sensitivity of ISC-EFS in the colon was the result of high cholinesterase activity because neostigmine revealed cholinergic components. Colonic ISC-EFS remained unchanged after tachykinin, P2X, P2Y or A1 and A2 receptor blockade. R-basal was smaller and ISC-basal was higher in the small intestine. TTX and bumetanide decreased ISC-basal in all regions, suggesting nerve-dependent secretory tone. ISC-basal was atropine-sensitive in the small intestine and PG97-269-sensitive in the large intestine. This comprehensive study reveals novel insights into region-specific nerve-mediated secretion in the human small and large intestine.

Chronic stress and peripheral pain: Evidence for distinct, region-specific changes in visceral and somatosensory pain regulatory pathways.

Chronic stress alters the hypothalamic-pituitary-adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6-S2 DRG but not L4-L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4-L5 but not L6-S2 DRGs in stressed rats, which was reproduced in control DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4-L5 DRGs normalized 3 days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord.

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung's Disease.

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung's disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung's disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung's patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.

Characterization of ghrelin-sensitive neurons in the lumbosacral defecation center in rats.

BACKGROUND: Ghrelin is involved in the regulation of somatic growth, feeding behavior and energy homeostasis. Ghrelin stimulates neuropeptide Y (NPY) neurons and activates intracellular AMP-activated protein kinase (AMPK) in the hypothalamus. These NPY neurons also express the leptin receptor and leptin inhibits ghrelin-induced activation of NPY neurons. In the spinal cord, we have demonstrated colokinetic action of ghrelin. However, the precise characteristics of the ghrelin-sensitive neurons remain to be clarified. The aim of this study was firstly to confirm that the action of ghrelin is mediated via a neurogenic pathway in the spinal cord, and secondly to characterize the ghrelin-sensitive neurons by comparing with hypothalamic ghrelin-sensitive neurons. METHODS: Rats were anesthetised with alpha-chloralose and ketamine, and colorectal intraluminal pressure and expelled volume were recorded in vivo. Drugs were applied intrathecally. KEY RESULTS: Ghrelin caused enhancement of propulsive contractions. Tetrodotoxin completely blocked the colokinetic effect of ghrelin. An AMPK activator, aminoimidazole carboxamide ribonucleotide, failed to mimic the ghrelin effect. Leptin had no effect on the spontaneous contractions and did not exert a suppressive effect on the ghrelin-enhanced colorectal motility. An NPY Y1 receptor antagonist did not affect the action of ghrelin. NPY had no effect on the colorectal motility. CONCLUSIONS & INFERENCES: This study showed that intrathecal injection of ghrelin stimulates colorectal motility by acting on ghrelin-sensitive neurons in the lumbosacral defecation center. The characteristics of ghrelin-sensitive neurons in the spinal cord are quite different from those of ghrelin-sensitive neurons in the hypothalamus.

Identification of different types of spinal afferent nerve endings that encode noxious and innocuous stimuli in the large intestine using a novel anterograde tracing technique.

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.

Organisation of autonomic nervous structures in the large intestine of chinchilla (Chinchilla laniger Molina).

The organization of autonomic nerve structures in the large intestine of chinchilla was investigated using histochemical and immunocytochemical methods. The myenteric plexus formed network nodes of cholinergic neurocyte agglomerations connected with bundles of nerve fibres and localized between the circular and longitudinal layers of the smooth muscles. The highest density of myenteric plexus was observed in the rectum. The different densities of myenteric plexus in subsequent parts of the large intestine is connected with the disparate functions of this part of the gut. The submucous plexus was distributed at several levels of mucosa and was a more dispersed structure than the myenteric plexus. Characteristic varicose adrenergic fibres were observed within the myenteric and submucous plexus in different layers of the large intestine wall.

Counting neurons is not as easy as 'one-two, three'.

An accurate determination of the number of neurons in a segment of bowel is fundamental to establish population norms and identify neurodegenerative conditions, including age-related loss of myenteric ganglion cells. Although the latter phenomenon has been observed by several laboratories in various mammals, in this issue of Neurogastroenterology and Motility, Gamage et al. present evidence that colonic myenteric ganglion cells are maintained in aged mice. These discordant findings prompt a thoughtful consideration, the range of variables affecting the accuracy of neuronal counts and the survival of neuronal populations in aging animals.

Physical and psychological effects of treatment on sexual functioning in colorectal cancer survivors.

INTRODUCTION: As a result of advances in surgical techniques and (neo)adjuvant therapy, mortality rates for colorectal cancer (CRC) have declined significantly in the last two decades. In general, CRC survivors report good health-related quality of life in survivorship. However, many survivors, including those who describe their quality of life as good, also report sexual problems that persist long after treatment is completed. AIMS: In this article, we review the effects of different treatment modalities for CRC on sexual functioning in men and women. We highlight both the physical and psychological aspects of CRC treatment and discuss the management of common sexual problems in CRC survivors. METHODS: The authors reviewed the existing available published articles regarding this topic. RESULTS: Our review of the evidence suggests that surgical treatment and (neo)adjuvant therapy for CRC are commonly associated with a wide range of sexual problems. CONCLUSIONS: Sexual functioning is an important functional outcome after CRC treatment and is influenced by a myriad of clinical and patient factors, including an individual's physical and psychological well-being after a diagnosis of CRC. As such, the assessment and management of sexual functioning in men and women with CRC should begin prior to the initiation of treatment and continue throughout treatment and survivorship.

Slit/Robo-mediated chemorepulsion of vagal sensory axons in the fetal gut.

BACKGROUND: The vagus nerve descends from the brain to the gut during fetal life to reach specific targets in the bowel wall. Vagal sensory axons have been shown to respond to the axon guidance molecule netrin and to its receptor, deleted in colorectal cancer (DCC). As there are regions of the gut wall into which vagal axons do and do not extend, it is likely that a combination of attractive and repellent cues are involved in how vagal axons reach specific targets. We tested the hypothesis that Slit/Robo chemorepulsion can contribute to the restriction of vagal sensory axons to specific targets in the gut wall. RESULTS: Transcripts encoding Robo1 and Robo2 were expressed in the nodose ganglia throughout development and mRNA encoding the Robo ligands Slit1, Slit2, and Slit3 were all found in the fetal and adult bowel. Slit2 protein was located in the outer gut mesenchyme in regions that partially overlap with the secretion of netrin-1. Neurites extending from explanted nodose ganglia were repelled by Slit2. CONCLUSIONS: These observations suggest that vagal sensory axons are responsive to Slit proteins and are thus repelled by Slits secreted in the gut wall and prevented from reaching inappropriate targets.

Ca(2+) transients in submucous neurons during the colonic migrating motor complex in the isolated murine large intestine.

BACKGROUND: The colonic migrating motor complex (CMMC) is a spontaneous, rhythmic, and neurally mediated motor pattern generated by myenteric neurons, which can propel fecal pellets in mice. Our aim was to determine whether submucous neurons were also activated during the CMMC. METHODS: :The isolated murine colon was opened and sections of mucosa were removed to expose the submucous ganglia, which were then loaded with Fluo-4. KEY RESULTS: Colonic migrating motor complexes, which occurred spontaneously or by mechanically stimulating the mucosa, were identified by displacement of the tissue (duration = 23.3 s). Between CMMCs, spontaneous Ca(2+) transients (frequency = 0.9 Hz) were observed in 55% (n = 8) of submucous neurons. During the CMMC, 98% (seven ganglia, n = 7) of submucous neurons within the same ganglion exhibited rapid Ca(2+) transients (1.6 Hz) superimposed on a sustained rise in Ca(2+) (duration ∼23 s) that occurred 1.7 s following the mucosal stimulus; whereas other neurons exhibited a similar, but delayed response that occurred either at 7 or 13 s following the stimulus. The activity in submucous neurons was correlated with activity in adjacent nerve varicosities. Ondansetron (1 mm; 5-HT(3) antagonist) significantly reduced the frequency and duration of the Ca(2+) transient responses. CONCLUSIONS & INFERENCES: Activity in the submucous neurons appears to be secondary to that in the myenteric plexus and appears to be generated largely by activity in myenteric descending (serotonergic) interneurons. During the CMMC, there is likely to be an increase in secretion to lubricate and facilitate fecal pellet propulsion.

Activity in varicosities within the myenteric plexus between and during the colonic migrating motor complex in the isolated murine large intestine.

BACKGROUND: Neuronal communication within the myenteric plexus occurs when action potentials along nerve fibers produce Ca(2+) transients in varicosities leading to exocytosis of vesicles and neurotransmitters release. We used Ca(2+) transients in varicosities to monitor action potential activity in myenteric nerve pathways both between and during the colonic migrating motor complex (CMMC) in the isolated murine colon. METHODS: Strips of longitudinal muscle were removed to reveal the myenteric ganglia, which were then loaded with Fluo-4. KEY RESULTS: Many varicosities, including synaptotagmin 1 labeled varicosities, exhibited ongoing Ca(2+) transients (duration of unitary Ca(2+) transient 3.9 s). Between CMMCs, varicosities fired at a frequency of 0.6 Hz, which correlated with spontaneous inhibitory junction potentials in the circular muscle, suggesting they were mainly in inhibitory nerve pathways. During a CMMC other previously quiescent varicosities fired at 1.3 Hz (max. 2.0 Hz) for the duration (24 s) of the CMMC, suggesting they were on excitatory nerve pathways. Activity in varicosities was correlated with Ca(2+) transient responses in a number of neurons. Some varicosities appeared to release an inhibitory neurotransmitter that reduced activity in nNOS-positive neurons. Varicosities along the same nerve fiber exhibited identical patterns of activity that allowed nerve fibers to be traced throughout the myenteric plexus and internodal strands. Activity in varicosities was reduced by hexamethonium (100 µmol L(-1) ), and blocked by ω-conotoxin GVIA (200 nM) and tetrodotoxin (1 µmol L(-1) ; TTX). CONCLUSIONS & INFERENCES: Ca(2+) imaging of varicosities allows for a determination of activity in neural pathways within the enteric nervous system.

Does colorectal endometriosis alter intestinal functions? A prospective manometric and questionnaire-based study.

OBJECTIVE: To objectively evaluate using anorectal manometry whether endometriotic nodules influence intestinal function and to reveal subjective intestinal dysfunctions in patients with rectosigmoid deep infiltrating endometriosis. DESIGN: Prospective study. SETTING: Tertiary care university hospital. PATIENT(S): Patients (n = 25) with a preoperative diagnosis of rectosigmoid endometriosis. INTERVENTION(S): Patients underwent anorectal manometry; after that, they filled a questionnaire about defecatory functions and ranked their pain symptoms. MAIN OUTCOME MEASURE(S): The parameters studied were resting pressure, maximum squeezing pressure, pushing, rectoanal inhibitory reflex, and rectal sensibility. We analyzed the responses to the defecatory function questionnaire and the scored the endometriosis pain symptoms using a Visual Analogue Scale. RESULT(S): No alterations of the rectoanal inhibitory reflex were found. Hypertone of the internal anal sphincter was found in 20 of 25 patients. Almost half of the patients had an increase of the threshold of desire to defecate, and 7 patients had a reduction of the anal sphincter squeeze pressure. According to the responses to the defecatory function questionnaire, incomplete evacuation was the most common symptom. CONCLUSION(S): We did not find marked motility or sensitive dysfunctions at the anorectal manometry, whereas subjectively patients reported some defecatory disorders. We revealed the presence of hypertone of the internal anal sphincter in most of the patients. CLINICAL TRIAL REGISTRATION NUMBER: 74/2010/O/Sper.