Excitability and Synaptic Transmission in the Enteric Nervous System: Does Diet Play a Role?

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

Detection of melatonin production from the intestinal epithelium using electrochemical methods.

The role of melatonin in the gastrointestinal (GI) tract had previously been limited to its well-described anti-oxidant properties. Recent studies have, however, expanded the role of melatonin in the intestine, showing that it acts as a hormone with local paracrine actions to modulate GI function and the release of other hormones. The GI epithelium produces melatonin from the active precursor serotonin, which is thought to come from the serotonin synthesising enterochromaffin cells (EC). The receptors for melatonin, the membrane bound melatonin receptors 1 and 2, are present on some smooth muscles, neurons, and epithelium. Endogenous release of melatonin has been linked with secretory reflexes and the ileal brake reflex, while exogenous application of melatonin in pharmacological doses has been associated with reduced inflammation in a variety of animal models. Recent studies have begun to look at melatonin release from the GI epithelium using real-time electrochemical detection methods. Using these techniques, the time course of melatonin production shows similarities to that of 5-HT release while the ratio of 5-HT to melatonin is altered during aging. In addition, the effects of melatonin supplementation on the production of endogenous melatonin and its precursor serotonin are suppressed. In summary, the role of melatonin in the GI tract is coming of age. There are many studies showing a clear role for endogenously produced melatonin and clear effects of melatonin supplementation. Newly developed electrochemical techniques for exploring melatonin availability in real-time promise to accelerate our understanding of GI melatonin in the years to come.

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity.

AIMS: Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. METHODS AND RESULTS: TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation. CONCLUSION: The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

Endothelium-dependent vasodilation in human mesenteric artery is primarily mediated by myoendothelial gap junctions intermediate conductance calcium-activated K+ channel and nitric oxide.

Myoendothelial microdomain signaling via localized calcium-activated potassium channel (K(Ca)) and gap junction connexins (Cx) is critical for endothelium-dependent vasodilation in rat mesenteric artery. The present study determines the relative contribution of NO and gap junction-K(Ca) mediated microdomain signaling to endothelium-dependent vasodilation in human mesenteric artery. The hypothesis tested was that such activity is due to NO and localized K(Ca) and Cx activity. In mesenteric arteries from intestinal surgery patients, endothelium-dependent vasodilation was characterized using pressure myography with pharmacological intervention. Vessel morphology was examined using immunohistochemical and ultrastructural techniques. In vessel segments at 80 mm Hg, the intermediate (I)K(Ca) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H-pyrazole (TRAM-34; 1 µM) inhibited bradykinin (0.1 nM-3 µM)-induced vasodilation, whereas the small (S) K(Ca) blocker apamin (50 and 100 nM) had no effect. Direct IK(Ca) activation with 1-ethyl-2-benzimidazolinone (1-EBIO; 10-300 µM) induced vasodilation, whereas cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (1-30 µM), the SK(Ca) activator, failed to dilate arteries, whereas dilation induced by 1-EBIO (10-100 µM) was blocked by TRAM-34. Bradykinin-mediated vasodilation was attenuated by putative gap junction block with carbenoxolone (100 µM), with remaining dilation blocked by N-nitro l-arginine methyl ester (100 µM) and [1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (10 µM), NO synthase and soluble guanylate cyclase blockers, respectively. In human mesenteric artery, myoendothelial gap junction and IK(Ca) activity are consistent with Cx37 and IK(Ca) microdomain expression and distribution. Data suggest that endothelium-dependent vasodilation is primarily mediated by NO, IK(Ca), and gap junction Cx37 in this vessel. Myoendothelial microdomain signaling sites are present in human mesenteric artery and are likely to contribute to endothelium-dependent vasodilation via a mechanism that is conserved between species.

Serotonin release and uptake in the gastrointestinal tract.

The afferent innervation of the gastrointestinal (GI) tract consists of intrinsic and extrinsic sensory neurons that respond to nutrients, chemicals or mechanical stimuli within the gut lumen. Most stimuli do not interact directly with the afferent nerves but instead activate specialised cells in the epithelium in a process of sensory transduction. It is thought that one of the first steps in this process is the release of serotonin (5-HT) from the enterochromaffin (EC) cells. The EC cells are a sub-type of enteroendocrine (EE) cells which are found among the enterocytes of the intestinal epithelium. The EC cells are responsible for the production and storage of the largest pool of 5 HT in the body. Released 5-HT can act on the intrinsic nerves and vagal endings. This review will focus on the role of 5-HT in sensory transduction and examine how the EC cell produces and releases 5-HT. We will explore recent developments that have helped to elucidate some of the proteins that allow EC cells to sense the luminal environment. Finally, we will highlight some of the findings from new studies using electrochemical techniques which allow the real-time recording of 5-HT concentrations near to the EC cell.

Analysis of real-time serotonin (5-HT) availability during experimental colitis in mouse.

Serotonin (5-HT)-containing enterochromaffin (EC) cells of the intestine transduce chemical and mechanical stimuli from the intestinal lumen by releasing 5-HT on to afferent nerve terminals. Dysfunctional mucosal 5-HT signaling has been implicated in heightened visceral sensitivity and altered motility in patients with inflammatory bowel disease and in animal models. Our aim was to characterize the release and uptake of 5-HT in the mouse dextran sulfate sodium (DSS; 5% wt/vol) model of colitis. We made electrochemical recordings and used an ELISA assay to determine mucosal 5-HT release and uptake in untreated mice and mice with DSS-induced colitis. Peak and steady-state 5-HT concentrations were measured before and during blockade of the serotonin reuptake transporter (SERT) with 1 microM fluoxetine. Electrochemical recordings showed that colons from DSS-treated mice had roughly twice the steady-state levels of extracellular 5-HT and compression-evoked 5-HT release compared with untreated mice. Fluoxetine doubled the compression-evoked and steady-state 5-HT levels in control and DSS mice. These data were supported by ELISA assays, which showed enhanced 5-HT release during colitis, by immunohistochemical analyses, which showed increases in EC cell numbers, and by real-time PCR, which identified a decrease in SERT mRNA expression in the mucosa during colitis. These data are the first to demonstrate 5-HT release close to its release site and near its site of action during DSS-colitis. We conclude that DSS-colitis increases 5-HT availability primarily by an increase in the numbers of EC cells and/or of content of 5-HT in these EC cells.

Serotonin (5-HT) release and uptake measured by real-time electrochemical techniques in the rat ileum.

Serotonin (5-HT) is released from the enterochromaffin cells and plays an important role in regulating intestinal function. Although the release of 5-HT is well documented, the contribution of the serotonin reuptake transporter (SERT) to the levels and actions of 5-HT in the intestine is unclear. This study aimed to demonstrate real-time SERT activity in ileal mucosa and to assess the effects of SERT inhibition using fluoxetine. Electrochemical recordings were made from the mucosa in full-thickness preparations of rat ileum using a carbon fiber electrode to measure 5-HT oxidation current and a force transducer to record circular muscle (CM) tension. Compression of the mucosa stimulated a peak 5-HT release of 12 +/- 6 microM, which decayed to 7 +/- 4 microM. Blockade of SERT with fluoxetine (1 microM) increased the peak compression-evoked release to 19 +/- 9 microM, and the background levels of 5-HT increased to 11 +/- 7 microM (P < 0.05, n = 7). When 5-HT was exogenously applied to the mucosa, fluoxetine caused a significant increase in the time to 50% and 80% decay of the oxidation current. Fluoxetine also increased the spontaneous CM motility (P < 0.05; n = 7) but did not increase the CM contraction-evoked 5-HT release (P > 0.05, n = 5). In conclusion, this is the first characterization of the real-time uptake of 5-HT into the rat intestine. These data suggest that SERT plays an important role in the modulation of 5-HT concentrations that reach intestinal 5-HT receptors.

Teaching basic gastrointestinal physiology using classic papers by Dr. Walter B. Cannon.

The movements of the gastrointestinal tract, as described by Walter B. Cannon 100 years ago, reveal much about the functions of this unique organ and how it is controlled by the body. Two classic papers by Cannon provide a rare glimpse into the hidden functions of the body and give students a great example of the scientific method in action. In this essay, we describe the basic movements and functions of the gastrointestinal tract as revealed by X-rays, contrast meals, and Cannon's careful observations. It is Cannon's experimental care and obvious interest in his subject matter that can provide your students with a taste of the excitement of discovery and insight into the processes by which experimental science moves forward.