Excitability Of Enteric Neurons Research Articles

Research advances in the mechanism of acupuncture and moxibustion in regulating gastrointestinal motility and related thinking

Gastrointestinal motility dysfunction is not only a pathogenic factor but also a pathological manifestation. The authors summarize the recent studies on the mechanism of acupuncture and moxibustion in regulating gastrointestinal motility and analyze the effect of acupuncture and moxibustion on the brain-vagus-enteric nervous system, interstitial cells of Cajal, gastrointestinal peptides, gastrointestinal smooth muscle, and signaling pathways. The authors also put forward some suggestions for future research, including the research centering on gastrointestinal motility to explore the role of acupuncture and moxibustion in regulating gastrointestinal motility for disease prevention and treatment, specificity of acupoints, and dual-directional regulation of acupuncture and moxibustion, and the research focusing on intestinal microflora and influential factors for excitability of enteric neurons to explore the potential mechanism of acupuncture and moxibustion in regulating gastrointestinal motility, in order to provide a reference for future research.

Tryptase potentiates enteric nerve activation by histamine and serotonin: Relevance for the effects of mucosal biopsy supernatants from irritable bowel syndrome patients.

We previously showed that mucosal biopsy supernatants from irritable bowel syndrome patients activated neurons despite low concentrations of tryptase, histamine, and serotonin which individually would not cause spike discharge. We studied the potentiating responses between these mediators on excitability of enteric neurons. Calcium-imaging was performed using the calcium-sensitive dye Fluo-4 AM in human submucous plexus preparations from 45 individuals. Histamine, serotonin, and tryptase were applied alone and in combinations to evaluate nerve activation which was assessed by analyzing increase in intracellular Ca2+ ([Ca2+ ]i ), the proportion of responding neurons and the product of both defined as Ca-neuroindex (NI). Protease activated receptor (PAR) 2 activating peptide, PAR2 antagonist and the serine protease-inhibitor FUT-175 were used to particularly investigate the role of proteases. Histamine or serotonin (1μmol/L each) evoked only few small responses (median NI [25%/75%]: 0 [0/148]; 85 [0/705] respectively). Their combined application evoked statistically similar responses (216 [21/651]). Addition of the PAR2 activator tryptase induced a significantly higher Ca-NI (1401 [867/4075]) compared to individual application of tryptase or to coapplied histamine and serotonin. This synergistic potentiation was neither mimicked by PAR2 activating peptide nor reversed by the PAR2 antagonist GB83, but abolished by FUT-175. We observed synergistic potentiation between histamine, serotonin, and tryptase in enteric neurons, which is mediated by proteolytic activity rather than PAR2 activation. This explained neuronal activation by a cocktail of these mediators despite their low concentrations and despite a relatively small PAR2-mediated response in human submucous neurons.

Ion channel expression in the developing enteric nervous system.

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

Effects of HIV‐1 tat protein on excitability of enteric neurons

HIV is mostly transmitted across mucosal surfaces including vaginal, anorectal and oro‐intestinal mucosa. Recent studies demonstrate that HIV infection affects gastrointestinal motility including diarrhea being a serious problem with significant effect on the quality of life. Trans‐activator of transcription (Tat) is an HIV gene that enhances the efficiency of HIV gene transcription. Tat protein together with GP120 has been shown to induce neuronal death, however, its effects on enteric neuronal excitability is not known. In this study we examined the effect of acute (0–30 min) as well as chronic (up to 48 hrs) administration of Tat (1–86) on the electrical activity of mouse enteric neurons in culture. In whole cell patch clamp studies under current clamp, Tat decreased the threshold for action potential generation by 6.8±0.7mV (n=4) within 10 mins of perfusion and increased the number of spikes for each individual neuron. This effect persisted in neurons pretreated with Tat for over 48 hrs (n=15). In some neurons, spontaneous action potentials were observed. The average resting membrane potential of enteric neurons in the presence of Tat was −49mV (n=15) and not significantly different for untreated cells (‐50mV; n= 15). These data suggest that Tat protein has a direct and persistent effect on neuronal excitability. Supported by NIH DA024009

Functions and Imaging of Mast Cell and Neural Axis of the Gut

Close association between nerves and mast cells in the gut wall provides the microanatomic basis for functional interactions between these elements, supporting the hypothesis that a mast cell-nerve axis influences gut functions in health and disease. Advanced morphology and imaging techniques are now available to assess structural and functional relationships of the mast cell-nerve axis in human gut tissues. Morphologic techniques including co-labeling of mast cells and nerves serve to evaluate changes in their densities and anatomic proximity. Calcium (Ca(++)) and potentiometric dye imaging provide novel insights into functions such as mast cell-nerve signaling in the human gut tissues. Such imaging promises to reveal new ionic or molecular targets to normalize nerve sensitization induced by mast cell hyperactivity or mast cell sensitization by neurogenic inflammatory pathways. These targets include proteinase-activated receptor (PAR) 1 or histamine receptors. In patients, optical imaging in the gut in vivo has the potential to identify neural structures and inflammation in vivo. The latter has some risks and potential of sampling error with a single biopsy. Techniques that image nerve fibers in the retina without the need for contrast agents (optical coherence tomography and full-field optical coherence microscopy) may be applied to study submucous neural plexus. Moreover, the combination of submucosal dissection, use of a fluorescent marker, and endoscopic confocal microscopy provides detailed imaging of myenteric neurons and smooth muscle cells in the muscularis propria. Studies of motility and functional gastrointestinal disorders would be feasible without the need for full-thickness biopsy.

The Nav1.9 channel regulates colonic motility in mice

The colonic migrating motor complex (CMMC) is a major pattern of motility that is entirely generated and organized by the enteric nervous system. We have previously demonstrated that the Nav1.9 channel underlies a tetrodotoxin-resistant sodium current which modulates the excitability of enteric neurons. The aim of this study was to observe the effect of loss of the Nav1.9 channel in enteric neurons on mouse colonic motility in vitro. The mechanical activity of the circular muscle was simultaneously recorded from three sites, namely, proximal, mid- and distal, along the whole colon of male, age-matched wild-type and Nav1.9 null mice. Spontaneous CMMCs were observed in all preparations. The mean frequency of CMMCs was significantly higher in the Nav1.9 null mice (one every 2.87 ± 0.1 min compared to one every 3.96 ± 0.23 min in the wild type). The mean duration of CMMCs was shorter and the mean area-under-contraction was larger in the Nav1.9 null mice compared to the wild type. In addition, CMMCs propagated preferentially in an aboral direction in the Nav1.9 null mice. Our study demonstrates that CMMCs do occur in mice lacking the Nav1.9 channel, but their characteristics are significantly different from controls. Up to now, the Nav1.9 channel was mainly associated with nociceptive neurons and involved in their hyperexcitability after inflammation. Our result shows for the first time a role for the Nav1.9 channel in a complex colonic motor pattern.

Morphine decreases neuronal excitability in mouse enteric neurons via alterations in Na+ channel kinetics

A major side‐effect of morphine and other opioids is severe constipation. Morphine decreases neurotransmitter release from myenteric neurons resulting in reduced peristalsis. In this study we used whole‐cell patch clamp techniques to directly study the effects of morphine on cell excitability and ionic currents in mouse enteric neurons. Primary cultures of mouse ileum myenteric neurons were prepared by enzymatic digestion from longitudinal muscle/myenteric plexus (LMMP) preparations and plated in modified Neurobasal A media. Two classes of neurons were identified based on the absence (12/33) and presence (21/33) of an after‐hyperpolarization (AHP) following evoked action potentials. In neurons with AHP, morphine (3 ÂμM) significantly reduced neuronal excitability via an increase in rheobase, a reduction in action potential height, and by inhibiting the neuron from firing multiple action potentials. The resting membrane potential (−51.8 mV) was not affected. The peak amplitude of Na+ currents were significantly attenuated by morphine. The availability of Na+ channels was markedly reduced by morphine as demonstrated by a shift in the steady‐state voltage‐dependent inactivation kinetics. These data demonstrate that inhibition of Na+ current results in reduced excitability of enteric neurons and contributes to the severe constipation concurrent with opioid administration. Supported by NIH DA024009.

Gut microbiota and sirtuins in obesity-related inflammation and bowel dysfunction

Obesity is a chronic disease characterized by persistent low-grade inflammation with alterations in gut motility. Motor abnormalities suggest that obesity has effects on the enteric nervous system (ENS), which controls virtually all gut functions. Recent studies have revealed that the gut microbiota can affect obesity and increase inflammatory tone by modulating mucosal barrier function. Furthermore, the observation that inflammatory conditions influence the excitability of enteric neurons may add to the gut dysfunction in obesity. In this article, we discuss recent advances in understanding the role of gut microbiota and inflammation in the pathogenesis of obesity and obesity-related gastrointestinal dysfunction. The potential contribution of sirtuins in protecting or regulating the circuitry of the ENS under inflamed states is also considered.

The anxiolytic effect of Bifidobacterium longum NCC3001 involves vagal pathways for gut-brain communication

The probiotic Bifidobacterium longum NCC3001 normalizes anxiety-like behavior and hippocampal brain derived neurotrophic factor (BDNF) in mice with infectious colitis. Using a model of chemical colitis we test whether the anxiolytic effect of B. longum involves vagal integrity, and changes in neural cell function. Methods Mice received dextran sodium sulfate (DSS, 3%) in drinking water during three 1-week cycles. Bifidobacterium longum or placebo were gavaged daily during the last cycle. Some mice underwent subdiaphragmatic vagotomy. Behavior was assessed by step-down test, inflammation by myeloperoxidase (MPO) activity and histology. BDNF mRNA was measured in neuroblastoma SH-SY5Y cells after incubation with sera from B. longum- or placebo-treated mice. The effect of B. longum on myenteric neuron excitability was measured using intracellular microelectrodes. Chronic colitis was associated with anxiety-like behavior, which was absent in previously vagotomized mice. B. longum normalized behavior but had no effect on MPO activity or histological scores. Its anxiolytic effect was absent in mice with established anxiety that were vagotomized before the third DSS cycle. B. longum metabolites did not affect BDNF mRNA expression in SH-SY5Y cells but decreased excitability of enteric neurons. In this colitis model, anxiety-like behavior is vagally mediated. The anxiolytic effect of B. longum requires vagal integrity but does not involve gut immuno-modulation or production of BDNF by neuronal cells. As B. longum decreases excitability of enteric neurons, it may signal to the central nervous system by activating vagal pathways at the level of the enteric nervous system.

Excitation of Enteric Neurons by Supernatants of Colonic Biopsies From Irritable Bowel Syndrome Patients (IBS) is Linked to Visceral Sensitivity

but DAT and NET contribute to 5-HT clearance. As the animals mature beyond 3 weeks, SERT function increases while DAT function declines. NET and SERT both contribute to 5-HT clearance in the mucosa of 6 week old guinea pigs. These data indicate that there are redundant transport mechanisms for 5-HT clearance in the gut mucosa. Monoamine transport inhibitors used to treat behavioral problems in pediatric patients could have unanticipated effects on gut function. (Supported by DK57039, DK082967, HD05197)

W1944 Expression of Channels Regulating Excitability of Enteric Neurons: RT-PCR Analysis of K V7 Channel and Na V1 α-Subunit mRNAs the Guinea-Pig Small Intestine

W1944 Expression of Channels Regulating Excitability of Enteric Neurons: RT-PCR Analysis of K V7 Channel and Na V1 α-Subunit mRNAs the Guinea-Pig Small Intestine

Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca2+-activated K+ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the alpha3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the alpha3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs.

Serotonin Excites Neurons in the Human Submucous Plexus via 5-HT 3 Receptors

Serotonin (5-hydroxytryptamine [5-HT]) is a key signaling molecule in the gut. Recently, the neural 5-HT3 receptor received a lot of attention as a possible target in functional bowel diseases. Yet, the 5-HT3 receptor-mediated changes in properties of human enteric neurons is unknown. We used a fast imaging technique in combination with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine to monitor directly the membrane potential changes in neurons of human submucous plexus from surgical specimens of 21 patients. An Ussing chamber technique was used to study 5-HT3 receptor involvement in chloride secretion. Local microejection of 5-HT directly onto ganglion cells resulted in a transient excitation of enteric neurons characterized by increased spike discharge. This response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, and blocked by the 5-HT3 receptor antagonist, tropisetron. The proportions of 5-HT-responsive nerve cells per ganglion ranged from 25.5% +/- 18.4% in the duodenum to 54.2% +/- 46.9% in the colon. Interestingly, 2-methyl-5-HT did not evoke chloride secretion in the human intestine but it did in the guinea-pig intestine. Specific 5-HT3A and 5-HT3B receptor subunit immunoreactivity as well as 5-HT3A and 5-HT3B receptor-specific messenger RNA were detected in the tissue samples. Based on co-labeling with the pan-neuronal marker HuC/D we conclude that submucous nerve cells potentially express heteromeric 5-HT3A/B receptors. We show that 5-HT excited human enteric neurons via 5-HT3 receptors, which may comprise both 5-HT3A and 5-HT3B receptor subunits.

Enteric nervous system, serotonin, and the irritable bowel syndrome

Intestinal motility, secretion, and blood flow are controlled and integrated by the enteric nervous system (ENS). The ENS is like a "brain-in-the-gut," with many of the neurophysiologic properties of the central nervous system. Serotonin is a neurotransmitter at synapses in the microcircuits of the ENS. Serotonin is also released from enterochromaffin cells and inflammatory/immune cells to act at serotonergic receptors on neurons of the ENS. Four important actions are (1) fast and (2) slow excitation of enteric neurons, (3) presynaptic inhibition of neurotransmitter release at synapses in ENS microcircuits, and (4) excitation of intestinal sensory afferent fibers. Fast excitation and stimulation of sensory afferents are mediated by 5-HT(3) serotonergic receptors and slow excitation by 5-HT(1P) receptors. Presynaptic inhibitory receptors are not conclusively defined. The efficacy of a new 5-HT(3) receptor blocking drug in the treatment of the diarrhea-predominant form of the irritable bowel syndrome in women suggests the importance of this receptor subtype in the mediation of neurogenic secretory diarrhea, motility abnormality, and abdominal pain and discomfort.