Search

In this chapter, we describe the role played by classical and alternative renin–angiotensin system (RAS) in the physiological regulation of gastrointestinal functions. RAS modulates gut motility and mucosal functions, including secretion, fluid, and nutrient absorption. We focus mainly on the activation of angiotensin II type 1 and 2 receptors located on the smooth muscle and epithelial cells or on the enteric neurons. Few studies indicate that alternative RAS may counteract classical RAS functions. Although data on the enteric RAS system are still scarce, they encourage further investigations in consideration also of a potential involvement in gastrointestinal disorders.

Hirschsprung disease is most often characterized by aganglionosis limited to the distal colon and rectum, and mice lacking the Endothelin receptor type B (Ednrb) faithfully recapitulate this phenotype. However, despite the presence of enteric ganglia in the small intestine, both human patients and Ednrb−/− mice suffer from dysmotility and altered gastrointestinal function, thus raising the possibility of enteric nervous system (ENS) abnormalities proximal to the aganglionic region. We undertook the present study to determine whether abnormalities with the ENS in ganglionated regions may account for abnormal gastrointestinal function. We performed single-cell RNA sequencing on ENS cells from the small intestine of Ednrb−/− mice and compared the results to a published single-cell dataset. Our results identified a missing population of neurons marked by the enzyme Gad2, which catalyzes the production of γ-Aminobutyric acid (GABA), in the small intestine of Ednrb−/− animals. This result was confirmed by immunostaining enteric ganglia from Ednrb−/− mice and their wild-type littermates. These data show for the first time that ganglionated regions of the Hirschsprung gut lack a neuronal subpopulation, which may explain the persistent gastrointestinal dysfunction after surgical correction of Hirschsprung disease.

An intact gut epithelium preserves the immunological exclusion of “non-self” entities in the external environment of the gut lumen. Nonetheless, information flows continuously across this interface, with the host immune, endocrine, and neural systems all involved in monitoring the luminal environment of the gut. Both pathogenic and commensal gastrointestinal (GI) bacteria can modulate centrally-regulated behaviors and brain neurochemistry and, although the vagus nerve has been implicated in the microbiota-gut-brain signaling axis, the cellular and molecular machinery that facilitates this communication is unclear. Studies were carried out in healthy Sprague–Dawley rats to understand cross-barrier communication in the absence of disease. A novel colonic-nerve electrophysiological technique was used to examine gut-to-brain vagal signaling by bacterial products. Calcium imaging and immunofluorescent labeling were used to explore the activation of colonic submucosal neurons by bacterial products. The findings demonstrate that the neuromodulatory molecule, glucagon-like peptide-1 (GLP-1), secreted by colonic enteroendocrine L-cells in response to the bacterial metabolite, indole, stimulated colonic vagal afferent activity. At a local level indole modified the sensitivity of submucosal neurons to GLP-1. These findings elucidate a cellular mechanism by which sensory L-cells act as cross-barrier signal transducers between microbial products in the gut lumen and the host peripheral nervous system.

Injury to the enteric nervous system (ENS) can cause several gastrointestinal (GI) disorders including achalasia, irritable bowel syndrome, and gastroparesis. Recently, a subpopulation of enteric glial cells with neuronal stem/progenitor properties (ENSCs) has been identified in the adult ENS. ENSCs have the ability of reconstituting the enteric neuronal pool after damage of the myenteric plexus. Since the estrogen receptor β (ERβ) is expressed in enteric glial cells and neurons, we investigated whether a selective ERβ agonist, LY3201, can influence neuronal and glial cell differentiation. Myenteric ganglia from the murine muscularis externa were isolated and cultured in either glial cell medium or neuronal medium. In glial cell medium, the number of glial progenitor cells (Sox10+) was increased by fourfold in the presence of LY3201. In the neuronal medium supplemented with an antimitotic agent to block glial cell proliferation, LY3201 elicited a 2.7-fold increase in the number of neurons (neurofilament+ or HuC/D+). In addition, the effect of LY3201 was evaluated in vivo in two murine models of enteric neuronal damage and loss, namely, high-fat diet and topical application of the cationic detergent benzalkonium chloride (BAC) on the intestinal serosa, respectively. In both models, treatment with LY3201 significantly increased the recovery of neurons after damage. Thus, LY3201 was able to stimulate glial-to-neuron cell differentiation in vitro and promoted neurogenesis in the damaged myenteric plexus in vivo. Overall, our study suggests that selective ERβ agonists may represent a therapeutic tool to treat patients suffering from GI disorders, caused by excessive neuronal/glial cell damage. ispartof: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA vol:115 issue:22 pages:5798-5803 ispartof: location:United States status: published

The enteric nervous system, often referred to as the second brain, is the largest assembly of neurons and glia outside the central nervous system. The enteric nervous system resides within the wall of the digestive tract and regulates local gut reflexes involved in gastrointestinal motility, blood flow and fluid transport; all these functions can be accomplished in the absence of the extrinsic innervation from the central nervous system. It is neurons and their circuitry within the enteric nervous system that govern the gut reflexes. However, it is becoming clear that enteric glial cells are also actively involved in this process through the bidirectional signaling with neurons and other cells in the gut wall. We synthesize the recently discovered modulatory roles of enteric gliotransmission in gut motility and provide our perspective for future lines of research.

The effects of quercetin supplementation in NADH-diaphorase positive (NADH-d) neurons of streptozotocin-induced diabetic rats was carried in this study. Fifteen male rats were divided into three groups: normoglycemic (N), diabetic (D) and diabetic supplemented with quercetin (DQ). Whole mount preparations of the muscular layer of the ileum underwent NADH-d histochemistry for evidencing the NADH-d neuronal subpopulation. Quantitative analyzes were performed on 30 random fields, and morphometric analyzes in 100 neuronal bodies and nuclei per animal. The supplementation promoted a 44 % reduction in the neuronal density in D group when compared to N group (p

Key points Tenascin X (TNX) functions in the extracellular matrix of skin and joints where it maintains correct intercellular connections and tissue architectureTNX is associated exclusively with vagal‐afferent endings and some myenteric neurones in mouse and human stomach, respectively.TNX‐deficient mice have accelerated gastric emptying and hypersensitivity of gastric vagal mechanoreceptors that can be normalized by an inhibitor of vagal‐afferent sensitivity.Cultured nodose ganglion neurones showed no changes in response to capsaicin, cholecystokinin and potassium chloride in TNX‐deficient mice.TNX‐deficient patients have upper gastric dysfunction consistent with those in a mouse model. Our translational studies suggest that abnormal gastric sensory function may explain the upper gut symptoms present in TNX deficient patients, thus making it important to study gastric physiology.TNX deficiency should be evaluated routinely in patients with connective tissue abnormalities, which will enable a better understanding of its role and allow targeted treatment. For example, inhibitors of vagal afferents‐baclofen could be beneficial in patients. These hypotheses need confirmation via targeted clinical trials. Abstract Tenascin‐X (TNX) is a glycoprotein that regulates tissue structure via anti‐adhesive interactions with collagen in the extracellular matrix. TNX deficiency causes a phenotype similar to hypermobility Ehlers–Danlos syndrome involving joint hypermobility, skin hyperelasticity, pain and gastrointestinal dysfunction. Previously, we have shown that TNX is required for neural control of the bowel by a specific subtype of mainly cholinergic enteric neurones and regulates sprouting and sensitivity of nociceptive sensory endings in mouse colon. These findings correlate with symptoms shown by TNX‐deficient patients and mice. We aimed to identify whether TNX is similarly present in neural structures found in mouse and human gastric tissue. We then determined whether TNX has a functional role, specifically in gastric motor and sensory function and nodose ganglia neurones. We report that TNX was present in calretinin‐immunoreactive extrinsic nerve endings in mouse and human stomach. TNX deficient mice had accelerated gastric emptying and markedly increased vagal afferent responses to gastric distension that could be rescued with GABAB receptor agonist. There were no changes in nodose ganglia excitability in TNX deficient mice, suggesting that vagal afferent responses are probably the result of altered peripheral mechanosensitivity. In TNXB‐deficient patients, significantly greater symptoms of reflux, indigestion and abdominal pain were reported. In the present study, we report the first role for TNX in gastric function. Further studies are required in TNX deficient patients to determine whether symptoms can be relieved using GABAB agonists., Key points Tenascin X (TNX) functions in the extracellular matrix of skin and joints where it maintains correct intercellular connections and tissue architectureTNX is associated exclusively with vagal‐afferent endings and some myenteric neurones in mouse and human stomach, respectively.TNX‐deficient mice have accelerated gastric emptying and hypersensitivity of gastric vagal mechanoreceptors that can be normalized by an inhibitor of vagal‐afferent sensitivity.Cultured nodose ganglion neurones showed no changes in response to capsaicin, cholecystokinin and potassium chloride in TNX‐deficient mice.TNX‐deficient patients have upper gastric dysfunction consistent with those in a mouse model. Our translational studies suggest that abnormal gastric sensory function may explain the upper gut symptoms present in TNX deficient patients, thus making it important to study gastric physiology.TNX deficiency should be evaluated routinely in patients with connective tissue abnormalities, which will enable a better understanding of its role and allow targeted treatment. For example, inhibitors of vagal afferents‐baclofen could be beneficial in patients. These hypotheses need confirmation via targeted clinical trials.

Present study aims to investigate the role of AGEs, TGF-β1, BDNF and their receptors on diabetes-induced colon remodeling. Diabetes was induced by a single tail vein injection 40 mg/kg of STZ. The parameters of morphometric and biomechanical properties of colonic segments were obtained from diabetic and normal rats. The expressions of AGE, RAGE, TGF- β1, TGF- β1 receptor, BDNF and TrkB were immunohistochemically detected in different layers of the colon. The expressions of AGE, RAGE, TGF-β1 and TGF- β1 receptor were increased whereas BDNF and TrkB were decreased in the diabetic colon (P

Promoter-based genetic recombination (via, e.g., Cre-lox) is most useful when all cells of interest express a particular gene. The discovery that the actin-binding protein advillin is expressed in all somatic sensory neurons has been exploited repeatedly to drive DNA recombination therein, yet specificity of expression has not been well demonstrated. Here, we characterize advillin expression amongst sensory neurons and in several other neural and non-neural tissues. We first validate an advillin antibody against advillin knock-out tissue, advillin promoter-driven EGFP, and advillin mRNA expression. In the dorsal root ganglion (DRG), advillin is enriched in non-peptidergic nociceptors. We also show that advillin expression, and advillin promotor-driven EGFP and Cre-recombinase expression, occurs in multiple tissues including the dorsal habenula of the epithalamus, endocrine cells of the gut, Merkel cells in the skin, and most strikingly, throughout the autonomic nervous system (sympathetic, parasympathetic, and enteric neurons) in mice, rats, and non-human primates. In the mouse pelvic ganglion, advillin immunoreactivity is most intense in pairs of small neurons, and concentrated in spine-like structures on the axon initial segment contacted by sympathetic preganglionic axons. In autonomic targets (iris and blood vessels), advillin is distributed along cholinergic parasympathetic axons and in sympathetic varicosities. Developmentally, advillin expression is absent from sympathetics at postnatal day 4 but begins to emerge by day 7, accounting for previous reports (based on embryonic expression) of advillin’s specificity to sensory neurons. These results indicate that caution is warranted in interpreting previous studies in which advillin-driven genomic editing is either constitutive or performed after postnatal day 4.

Background/Aims: Hirschsprung's disease (HSCR), known as aganglionosis, is an infrequent congenital gut motility disorder characterized by absence of enteric neurons. In this study, we focus on the role of the intronic miR-215 and its host gene isoleucyl-tRNA synthetase 2 (IARS2) in the pathogenesis of HSCR. Methods: Quantitative real time PCR and Western blot were used to detect the miRNA, mRNAs, and proteins levels. The dual-luciferase reporter gene assay confirmed the direct regulation of the specific mRNA and miRNAs in cell lines. Transwell assays, CCK8 assay, and flow cytometry were used to measure cell function of the human 293T and SH-SY5Y cells. Results: Expression levels of miR-215 in HSCR patient colon tissues were outstandingly lower than controls, which was positively correlated with expression of the host gene IARS2 and negatively correlated with predicted target gene: sialic acid binding Ig-like lectin 8 (SIGLEC-8). The loss of miR-215 inhibited cell migration and proliferation, which was consistent with the reduction of IARS2. The dual-luciferase reporter gene assay confirmed that miR-215 resulted in the inhibition of SIGLEC-8 by directly binding to the 3'-UTR of SIGLEC-8. Moreover, knocking-down SIGLEC-8 rescued the extent of suppressed cell migration and proliferation that resulted from the diminishment of miR-215. Conclusions: Our findings indicate that miR-215 acts in concert with the host gene IARS2 to affect neuron migration and proliferation through the target gene SIGLEC-8. We infer that the IARS2-miR-215-SIGLEC-8 pathway may play a crucial role in the pathogenesis of HSCR.

According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.

Voltage-gated sodium (Nav) channels play a central role in gastrointestinal physiology because they transmit depolarizing impulses in enteric neurons, thereby enabling the coordination of intestinal motility. However, little is known about the ion channel machinery that specifies firing pattern of enteric neurons. Here, we usedin situpatch-clamp recording of myenteric neurons from mice to define functionally the Nav channel subtypes responsible for the electrical signature of myenteric neurons. We found that mouse myenteric neurons exhibit two types of tetrodotoxin-resistant Na+currents: an early inactivating Na+current (INaT) and a persistent Na+current (INaP).INaTwas encountered in all myenteric neurons, whereasINaPwas preferentially found in Dogiel type II sensory neurons. Knock-out mouse studies, in combination with pharmacological assays, indicate thatINaTis carried by theScn5a-encoded “cardiac” Nav1.5, whereasINaPis attributed to theScn11a-encoded Nav1.9. Current-clamp experiments show that Nav1.9 flows at subthreshold voltages, generating tonic firing. In addition, action potential (AP) clamp reveals that Nav1.5 contributes to the upstroke velocity of APs, whereas Nav1.9, which remains active during the falling phase, opposes AP repolarization. We developed a computational model of a Dogiel type II myenteric neuron that successfully reproduces all experimentally observed phenomena and highlights the differential roles of Nav1.5 and Nav1.9 in the control of excitability. Our data illustrate how excitability can be finely tuned to provide specific firing templates by the selective deployment of Nav1.5 and Nav1.9 isoforms. We propose that Nav-dependent ENS disorders of excitability may play important roles in the pathogenesis of digestive diseases.

Leptin ameliorates the prosecretory and prokinetic effects of the pro-inflammatory cytokine interleukin-6 on rat colon. Leptin also suppresses the neurostimulatory effects of irritable bowel syndrome plasma, which has elevated concentrations of interleukin-6, on enteric neurons. This may indicate a regulatory role for leptin in immune-mediated bowel dysfunction. In addition to its role in regulating energy homeostasis, the adipokine leptin modifies gastrointestinal (GI) function. Indeed, leptin-resistant obese humans and leptin-deficient obese mice exhibit altered GI motility. In the functional GI disorder irritable bowel syndrome (IBS), circulating leptin concentrations are reported to differ from those of healthy control subjects. Additionally, IBS patients display altered cytokine profiles, including elevated circulating concentrations of the pro-inflammatory cytokine interleukin-6 (IL-6), which bears structural homology and similarities in intracellular signalling to leptin. This study aimed to investigate interactions between leptin and IL-6 in colonic neurons and their possible contribution to IBS pathophysiology. The functional effects of leptin and IL-6 on colonic contractility and absorptosecretory function were assessed in organ baths and Ussing chambers in Sprague–Dawley rat colon. Calcium imaging and immunohistochemical techniques were used to investigate the neural regulation of GI function by these signalling molecules. Our findings provide a neuromodulatory role for leptin in submucosal neurons, where it inhibited the stimulatory effects of IL-6. Functionally, this translated to suppression of IL-6-evoked potentiation of veratridine-induced secretory currents. Leptin also attenuated IL-6-induced colonic contractions, although it had little direct effect on myenteric neurons. Calcium responses evoked by IBS plasma in both myenteric and submucosal neurons were also suppressed by leptin, possibly through interactions with IL-6, which is elevated in IBS plasma. As leptin has the capacity to ameliorate the neurostimulatory effects of soluble mediators in IBS plasma and modulated IL-6-evoked changes in bowel function, leptin may have a role in immune-mediated bowel dysfunction in IBS patients.

The effects of protein malnutrition on the quantitative aspects of the myenteric plexus in the ileum of adult Rattus norvegicus were assessed. Thirty 90-day-old rats were divided into two groups: Control Group (CG, n=15) and Experimental Group (EG, n=15). The CG received 26% protein chow and the EG received 4% protein chow for 90 days. At the end of the experiment, the animals from the CG weighed 369.63±26.33, and the ones from the EG 215.34±56.31. The ileum was submitted to Giemsa, NADH- and NADPH-diaphorase technique in order to evidence nervous cells in the whole-mount preparations. Animals from the EG presented a 41.75% body weight loss in relation to the CG as well as 17.6% length reduction for the ileum-jejunum. Moreover, the organ was 41% lighter for the EG. Giemsa-stained neurons were 17.02% more concentrated in the EG (p>0.05). NADH-diaphorase-stained neurons were 26.6% more concentrated in the EG (p0,05). Os neurônios NADH-diaforase apresentaram-se 26,60% mais concentrados no GE (p

Two-photon microscopy (2PM) can enable high-resolution deep imaging of thick tissue by exciting a fluorescent dye and protein at anastomotic sites in the mouse small intestine in vivo. We performed gut surgery and transplanted neural stem cells (NSC) from the embryonic central nervous system after marking them with the fluorescent cell linker, PKH26. We found that neurons differentiated from transplanted NSC (PKH [+]) and newborn enteric neurons differentiated from mobilized (host) NSC (YFP [+]) could be localized within the granulation tissue of anastomoses. A 5-HT4-receptor agonist, mosapride citrate (MOS), significantly increased the number of PKH (+) and YFP (+) neurons by 2.5-fold (P

Background Patients receiving anticancer chemotherapy experience a multitude of gastrointestinal side-effects. However, the causes of these symptoms are uncertain and whether these therapeutics directly affect the enteric nervous system is unknown. Our aim was to determine whether the function and morphology of myenteric neurons are altered in specimens of the colon from chemotherapy-treated patients. Methods Colon specimens were compared from chemotherapy-treated and non-treated patients following colorectal resections for removal of carcinoma. Intracellular electrophysiological recordings from myenteric neurons and immunohistochemistry were performed in whole mount preparations. Key Results Myenteric S neurons from chemotherapy-treated patients were hyperexcitable; more action potentials (11.4 ± 9.4, p < 0.05) were fired in response to depolarising current pulses than in non-treated patients (1.4 ± 0.5). The rheobase and the threshold to evoke action potentials were significantly lower for neurons from chemotherapy-treated patients compared to neurons from non-treated patients (p < 0.01). Fast excitatory postsynaptic potential reversal potential was more positive in neurons from chemotherapy-treated patients (p < 0.05). An increase in the number of neurons with translocation of Hu protein from the cytoplasm to the nucleus was observed in specimens from chemotherapy-treated patients (103 ± 25 neurons/mm2, 37.2 ± 7.0%, n = 8) compared to non-treated (26 ± 5 neurons/mm2, 11.9 ± 2.7%, n = 12, p < 0.01). An increase in the soma size of neuronal nitric oxide synthase-immunoreactive neurons was also observed in these specimens. Conclusions & Inferences This is the first study suggesting functional and structural changes in human myenteric neurons in specimens of colon from patients receiving anticancer chemotherapy. These changes may contribute to the causation of gastrointestinal symptoms experienced by chemotherapy-treated patients.

Summary Slow-transit constipation is usually considered a colonic motor disorder. However, there is some evidence that abnormalities may be present in locations other than the colon. In particular, several studies have reported abnormal motor activity of the small bowel in these patients. We evaluated the neuropathological aspects of the terminal ileum in patients with slow-transit constipation to see whether abnormalities are present that may explain an abnormal motility of the small intestine. Specimens of the terminal ileum were obtained from 16 female patients (age range, 42-76 years) with slow-transit constipation undergoing surgery for intractable symptoms. Fifteen age- and sex-matched controls were used for comparison. Histologic and immunohistochemical evaluation of the myenteric plexus and the smooth muscle of the proximal ileal resection margin was carried out by means of hematoxylin and eosin, trichrome and periodic acid–Schiff stain, neuron-specific enolase, S-100, CD117, CD34, anti– α -actin, desmin, and vimentin antibodies. The patient group displayed a significantly reduced number of glial cells, compared with controls, in both the submucosal and the myenteric plexus. Only 1 of the 3 populations of interstitial cells of Cajal (that associated with the deep muscular plexus) was decreased in patients. No differences were found between patients and controls concerning ganglia neurons, fibroblast-like cells, enteric neurons, apoptotic phenomena, and smooth muscle. Patients with slow-transit constipation display neuropathological abnormalities of the terminal ileum to a lesser extent than those we previously found in the colon, which might explain the abnormal motor aspects sometimes found in these patients.

The tachykinin substance P (SP) acts on the gut muscle coat via its preferred receptor, neurokinin 1 (NK1r). In the mouse ileum, NK1r-immunoreactivity (NK1r-IR) was detected in neurons, in the interstitial cells of Cajal at the deep muscular plexus (ICC-DMP) and the myoid cells of the villi. SP-IR was detected in neurons and varicose nerve fibers, which were especially numerous at the DMP and closely associated with the ICC-DMP. In mice with a mutation in the W locus (c-kit mutant animals), innervation is suggested to be normal although few studies have actually tested this hypothesis. Indeed, studies demonstrating ICC-DMP integrity are lacking and whether SP- and NK1r-IR are normal in these animals has not been investigated. Our aim was to perform an immunohistochemical study on the ileum of a strain of heterozygous mice with a mutation in the W locus, the We/+ mice, to test this hypothesis. SP-IR nerve fibers were significantly more numerous than in wild type mice; NK1r-IR was clustered on the plasma membrane and also intracytoplasmatic in the neurons, but absent in the ICC-DMP. The richness in SP-IR nerve fibers and the NK1r-IR distribution in the neurons, similar to that of activated cells, might be attempts to compensate for the SP preferred receptor absence at the ICC-DMP. In conclusion, SP content and NK1r expression are noticeably different in c-kit mutants with respect to wild type mice, and probably causing an anomalous tachykininergic control of intestinal motility. Physiological studies on W mutant mice have to take into account that innervation in this animal model is affected by the c-kit mutation.

We have studied the morphological and quantitative aspects of the myenteric plexus neurons of the proximal colon in rats (Rattus norvegicus of Wistar strain) submitted to a protein deprivation during prenatal and lactation periods. Twenty pregnant dams were divided in four groups labeled according to the kind of nourishment they were given: Group NN, normal diet; Group DN, low protein diet during prenatal period, and normal diet during lactation period; Group ND, normal diet during prenatal period, and low protein diet during lactation period; Group DD, low protein diet during prenatal and lactation periods. Histological analyses were developed with proximal colon segments using the haematoxylin and eosin staining method. Membrane preparations were stained by Giemsa's method. The statistical analysis has demonstrated no significant difference among the means of neurons found in the four studied groups. It was noticed that the animals under protein deprivation during prenatal and lactation periods presented greater quantity of large and strongly basophilic myenteric neurons. This suggests that neurons have accumulated protein in the cytoplasm. O objetivo deste trabalho foi estudar os aspectos morfológicos e quantitativos dos neurônios do plexo mientérico do colo proximal de ratos Wistar submetidos a desnutrição protéica, durante a gestação e lactação. Foram utilizados vinte animais, denominados de acordo com o período em que suas mães receberam ração com baixo teor de proteína: grupo NN (normoalimentados), grupo DN (ração hipoprotéica na gestação e normal na lactação), grupo ND (ração normal na gestação e hipoprotéica na lactação) e grupo DD (ração hipoprotéica na gestação e lactação). Amostras do colo proximal destinaram-se a cortes histológicos, corados pela hematoxilina-eosina e a preparados de membrana corados pelo método de Giemsa. O tratamento estatístico não demonstrou diferença significativa entre as médias referentes ao número de neurônios encontrados nos diferentes grupos nutricionais. Verificou-se que animais que sofreram deprivação protéica nos períodos de gestação e lactação apresentaram maior proporção de neurônios mientéricos grandes e fortemente basofílicos, indicando portanto que estes acumularam material protéico em seu citoplasma.

Gastrointestinal motility involves interactions between myogenic and neurogenic processes intrinsic to the gut wall. We have compared the presence of propagating myogenic contractions of the isolated colon in four experimental animals (guinea pig, mouse, rabbit, and rat), following blockade of enteric neural activity. Isolated colonic preparations were distended with fluid, with the anal end either closed or open. Spatiotemporal maps of changes in diameter were constructed from video recordings. Distension-induced peristaltic contractions were abolished by tetrodotoxin (TTX; 0.6 μM) in all animal species. Subsequent addition of carbachol (0.1–1 μM) did not evoke myogenic motor patterns in the mouse or guinea pig, although some activity was observed in rabbit and rat colon. These myogenic contractions propagated both orally and anally and differed from neurogenic propagating contractions in their frequency, extent of propagation, and polarity. Niflumic acid (300 μM), used to block myogenic activity, also blocked neural peristalsis and thus cannot be used to discriminate between these mechanisms. In all species, except the mouse colon, small myogenic “ripple” contractions were revealed in TTX, but in both rat and rabbit an additional, higher-frequency ripple-type contraction was superimposed. Following blockade of enteric nerve function, a muscarinic agonist can evoke propulsive myogenic peristaltic contractions in isolated rabbit and rat colon, but not in guinea pig or mouse colon. Marked differences between species exist in the ability of myogenic mechanisms to propel luminal content, but in all species there is normally a complex interplay between neurogenic and myogenic processes.

Leuprolide is a gonadotropin-releasing hormone (GnRH) analog which has been shown to reduce symptoms in patients with irritable bowel syndrome (IBS) and chronic intestinal pseudo-obstruction (CIPO). The mechanism is not known, but one hypothesis is through down-modulation of luteinizing hormone (LH) secretion, a hormone whith antagonistic effect on gastrointestinal motility. However, presence of LH receptors in the gastrointestinal tract has never been described. The aim of this study was to find one possible way of action for leuprolide by examining the presence of the LH receptor, and if present, to see whether there was different expression in patients with or without dysmotility. Full-thickness biopsies from the bowel wall of patients with and without severe dysmotility were examined using immunohistochemistry staining. Biopsies showed expression of LH receptors on myenteric neurons and in glial cells, neutrophils, endothelial cells and mast cells. There was no difference in expression between patient groups.

Chronic constipation is still considered a functional or idiopathic disorder. However, in recent years there is evidence that its pathopysiological grounds may be actually due to a complex system of abnormalities of the enteric nervous system of these patients. In particular, as reported in this review, the enteric glial cells seem to be constantly involved in constipated patients, suggesting that (at least some forms of) constipation should be considered as true neuro-gliopathies.

The development of IBS symptoms – altered bowel function and abdominal cramping in a subset of adult subjects exposed to severe enteric infections opened up an unprecedented opportunity to understand the etiology of this poorly understood disorder. Perhaps, for the reasons that these symptoms follow a severe enteric infection, and mucosal biopsy tissues are readily available, the focus of most studies thus far has been to show that mild/low-grade mucosal inflammation persisting after the initial infection has subsided causes the IBS symptoms. Parallel studies in non-infectious IBS patients, who did not have prior enteritis, showed similar mild mucosal inflammation. Together, these studies examined the mucosal infiltration of specific immune cells, increase of select inflammatory mediators, mast cell and enterochromaffin cell hyperplasia, and epithelial permeability. In spite of the fact that the data on these topics were not consistent among different studies and clinical trials with prednisone, fluoxetine, and ketotifen failed to provide relief of IBS symptoms, the predominant conclusions were that mild mucosal inflammation is the cause of IBS symptoms. However, the circular smooth muscle cells, and myenteric neurons are the primary regulators of gut motility function, while primary afferent neurons and CNS play essential roles in induction of visceral hypersensitivity – no explanation was provided as to how mild mucosal inflammation causes dysfunction in cells far removed. Accumulating evidence shows that mild mucosal inflammation in IBS patients is in physiological range. It has little deleterious effects on cells within its own environment and therefore it is unlikely to affect cells in the muscularis externa. This review discusses the disconnect between the focus on mild/low-grade mucosal inflammation and the potential mechanisms and molecular dysfunctions in smooth muscle cells, myenteric neurons, and primary afferent neurons that may underlie IBS symptoms.

A novel class of enteric neurons projecting directly from the rectal wall to the spinal cord, "rectospinal neurons", was investigated in rats by combined retrograde neuronal tracing, immunocytochemistry and electron microscopy. Rectospinal neurons were almost confined to myenteric ganglia of the distal rectum below the pelvic diaphragm and were labeled preferentially by injections into spinal cord segments L6/S1. Injections into more rostral spinal cord segments resulted in hardly any labeled enteric neurons. Dorsal and ventral rhizotomy experiments indicated an almost exclusive projection of rectospinal neurons through dorsal roots L6/S1 to the respective spinal cord segments. Among various peptides immunostained, vasoactive intestinal polypeptide and calcitonin gene-related peptide were selectively found in rectospinal neurons, which were also shown to contain calbindin, neurofilament protein- and peripherin-immunoreactivity. Vasoactive intestinal polypeptide- and calbindin-immunostaining were frequently co-localized in the same perikarya, while calcitonin gene-related peptide-immunoreactive rectospinal neurons probably represented a separate population. Neonatal capsaicin treatment did not significantly reduce the number of rectospinal neurons. Electron microscopy revealed synaptic contacts on the surface of rectospinal neurons. Taken together, these results establish rectospinal neurons as an anatomically and neurochemically distinct class of enteric neurons. Synaptic contacts on rectospinal neurons suggest that these neurons may function as a direct link from the enteric to the central nervous system, thus indicating that connections between these two networks are reciprocal.

Patients with Parkinson’s disease develop motor disturbances often accompanied by peripheral autonomic dysfunctions, including gastrointestinal disorders, such as dysphagia, gastric stasis and constipation. While the mechanisms subserving enteric autonomic dysfunctions are not clearly understood, they may involve the enteric dopaminergic and/or nitrergic systems. In the present study, we demonstrate that rats with unilateral 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons develop a marked inhibition of propulsive activity compared to sham-operated controls, as indicated by a 60% reduction of daily fecal output at the 4th week of observation. Immunohistochemical data revealed that 6- hydroxydopamine treatment did not affect the total number of HuC/D-positive myenteric neurons in both the proximal and distal segments of ileum and colon. Conversely, in the distal ileum and proximal colon the number of nitrergic neurons was significantly reduced. These results suggest that a disturbed distal gut transit, reminiscent of constipation in the clinical setting, may occur as a consequence of a reduced propulsive motility, likely due to an impairment of a nitric oxide-mediated descending inhibition during peristalsis.

Chronic administration of morphine results in the development of tolerance to the analgesic effects and to inhibition of upper gastrointestinal motility but not to colonic motility, resulting in persistent constipation. In this study we examined the effect of chronic morphine in myenteric neurons from the adult mouse colon. Similar to the ileum, distinct neuronal populations exhibiting afterhyperpolarization (AHP)‐positive and AHP‐negative neurons were identified in the colon. Acute morphine (3 μM) decreased the number of action potentials, and increased the threshold for action potential generation indicative of reduced excitability in AHP‐positive neurons. In neurons from the ileum of mice that were rendered antinociceptive tolerant by morphine‐pellet implantation for 5 days, the opioid antagonist naloxone precipitated withdrawal as evidenced by increased neuronal excitability. Overnight incubation of ileum neurons with morphine also resulted in enhanced excitability to naloxone. Colonic neurons exposed to long‐term morphine, remained unresponsive to naloxone suggesting that precipitated withdrawal does not occur in colonic neurons. However, morphine‐treated colonic neurons from β‐arrestin2 knockout mice demonstrated increased excitability upon treatment with naloxone as assessed by change in rheobase, number of action potentials and input resistance. These data suggest that similar to the ileum, acute exposure to morphine in colonic neurons results in reduced excitability due to inhibition of sodium currents. However, unlike the ileum, dependence to chronic exposure of morphine develops in colonic neurons from the β‐arrestin2 knockout mice. These studies corroborate the in‐vivo findings of the differential role of neuronal β‐arrestin2 in the development of morphine tolerance/dependence in the ileum and colon., Tolerance to chronic morphine does not develop in the colon, resulting in persistent constipation. Here, we examined electrophysiological characteristics of colonic neurons isolated from adult mouse and find that precipitated withdrawal occurs in neurons from the ileum but not colon. However, enhanced neuronal excitability in colonic neurons reflecting dependence to morphine occurs in colonic neurons of β ‐arrestin2 knockout mice. These studies show that mechanisms of morphine dependence–like tolerance differ between the ileum and colon and require downregulation of β ‐arrestin2.