Rachel M. Gwynne

Transmission between neurons in the extensive enteric neural networks of the gut involves synaptic potentials with vastly different time courses and underlying conductances. Most enteric neurons exhibit fast excitatory post-synaptic potentials (EPSPs) lasting 20–50 ms, but many also exhibit slow EPSPs that last up to 100 s. When large enough, slow EPSPs excite action potentials at the start of the slow depolarization, but how they affect action potentials evoked by fast EPSPs is unknown. Furthermore, two other sources of synaptic depolarization probably occur in enteric circuits, activated via GABA_A or GABA_C receptors; how these interact with other synaptic depolarizations is also unclear. We built a compartmental model of enteric neurons incorporating realistic voltage-dependent ion channels, then simulated fast EPSPs, slow EPSPs and GABA_A or GABA_C ligand-gated Cl^- channels to explore these interactions. Model predictions were tested by imaging Ca²⁺ transients in myenteric neurons ex vivo as an indicator of their activity during synaptic interactions. The model could mimic firing of myenteric neurons in mouse colon evoked by depolarizing current during intracellular recording and the fast and slow EPSPs in these neurons. Subthreshold fast EPSPs evoked spikes during the rising phase of a slow EPSP, but suprathreshold fast EPSPs could not evoke spikes later in a slow EPSP. This predicted inhibition was confirmed by Ca²⁺ imaging in which stimuli that evoke slow EPSPs suppressed activity evoked by fast EPSPs in many myenteric neurons. The model also predicted that synchronous activation of GABA_A receptors and fast EPSPs potentiated firing evoked by the latter, while synchronous activation of GABA_C receptors with fast EPSPs, potentiated firing and then suppressed it. The results reveal that so-called slow EPSPs have a biphasic effect being likely to suppress fast EPSP evoked firing over very long periods, perhaps accounting for prolonged quiescent periods seen in enteric motor patterns.

The prokinetic effects of 5‐HT₄ receptor (5‐HT₄R) agonists have been utilized clinically for almost three decades to relieve symptoms of constipation. Surprisingly, the mechanism(s) of action of these compounds is still being debated. Recent studies highlight luminal 5‐HT₄Rs as an alternative and effective target for these prokinetic agents. These include the study by Shokrollahi et al (2019, Neurogastroenterol Motil, e13598) published in the current issue of Neurogastroenterology and Motility, who found that activation of mucosal 5‐HT₄Rs by intraluminal prucalopride, significantly enhanced propulsive motor patterns in rabbit colon. The authors highlight the idea that development of agonists targeting luminal 5‐HT₄Rs in the colonic mucosa might be more effective and safer in achieving prokinetic effects on intestinal motility. The purpose of this mini‐review is to discuss the evidence for luminal 5‐HT₄Rs as an emerging target for prokinetic agents in facilitating propulsive motor patterns in the colon.

Amino acids applied to the mucosa evoke inhibitory reflexes in guinea-pig jejunum, but the receptors involved in sensory transduction are still unclear. One promising candidate is the extracellular calcium sensing receptor (CaSR), which is expressed by mucosal enteroendocrine cells and is preferentially activated by aromatic L-amino acids. We tested this by applying various amino acids to the mucosa and recording the resulting inhibitory junction potentials (IJPs) in nearby circular smooth muscle via intracellular recording. The CaSR is stereospecific and L-Phenylalanine evoked a significantly larger response than D-Phenylalanine when both were applied to the same site. The same pattern was seen with L- and D-Tryptophan, another aromatic amino acid. The CaSR is preferentially activated by aromatic amino acids and responses to L-Leucine and L-Lysine were significantly lower than those to L-Phenylalanine applied to the same site. Responses to L-Phenylalanine were dose-dependently suppressed by blockade of the CaSR with NPS2143, a CaSR antagonist, and mimicked by mucosal application of cinacalcet, a CaSR agonist. Responses to cinacalcet had similar pharmacology to that of responses to L-Phenylalanine, in that each requires both P2 purinoreceptors and 5-HT receptors. L-Glutamate evoked IJPs similar to those produced by L-Phenylalanine and these were depressed by blockade of P2 receptors and 5-HT₃ plus 5-HT₄ receptors, but NPS2143 was ineffective. The AMPA receptor antagonists DNQX (10 μM) and CNQX (10 μM) reduced IJPs evoked by L-Glutamate by 88 and 79% respectively, but neither BAY367260 (mGluR5 antagonist) nor 2APV (NMDA antagonist) affected IJPs evoked by L-Glutamate. We conclude that local inhibitory reflexes evoked by aromatic L-amino acids in guinea pig jejunum involve activation of CaSRs which triggers release of ATP and 5-HT from the mucosa. L-Glutamate also evokes inhibitory reflexes, via a pathway that does not involve CaSRs. It is likely there are multiple receptors acting as sensory transducers for different luminal amino acids.

Background & Aims: Cholera toxin (CT)-induced hypersecretion requires activation of secretomotor pathways in the enteric nervous system (ENS). AH neurons, which have been identified as a population of intrinsic sensory neurons, are a source of excitatory input to the secretomotor pathways. We therefore examined effects of CT in the intestinal lumen on myenteric and submucosal AH neurons. Methods: Isolated segments of guinea pig jejunum were incubated for 90 min with saline plus CT (12.5 µg/ml) or CT + neurotransmitter antagonist, or CT + TTX in their lumen. After washing CT away, submucosal or myenteric plexus preparations were dissected keeping circumferentially adjacent mucosa intact. Submucosal AH neurons were impaled adjacent to intact mucosa and myenteric AH neurons were impaled adjacent to, more than 5 mm from, and in the absence of intact mucosa. Neuronal excitability was monitored by injecting 500 ms current pulses through the recording electrode. Results: After CT pre-treatment, excitability of myenteric AH neurons adjacent to intact mucosa (n=29) was greater than that of control neurons (n=24), but submucosal AH neurons (n=33, control n=27) were unaffected. CT also induced excitability increases in myenteric AH neurons impaled distant from the mucosa (n=6) or in its absence (n=5). Coincubation with tetrodotoxin or SR142801 (NK3 receptor antagonist), but not SR140333 (NK1 antagonist) or granisetron (5-HT3 receptor antagonist) prevented the increased excitability induced by CT. Increased excitability was associated with a reduction in the characteristic AHP and an increase in the ADP of these neurons, but not a change in the hyperpolarisation-activated inward current in Ih. Conclusions: CT increases excitability of myenteric, but not submucosal, AH neurons. This is neurally mediated and depends on NK3, but not 5-HT3 receptors. Therefore, CT may act to amplify the secretomotor response to CT via an increase in the activity of the afferent limb of the enteric reflex circuitry.

Background and Purpose: Oxaliplatin is a platinum‐based chemotherapeutic drug used as a first‐line therapy for colorectal cancer. However, its use is associated with severe gastrointestinal side‐effects resulting in dose limitations and/or cessation of treatment. In this study, we tested whether oxidative stress, caused by chronic oxaliplatin treatment, induces enteric neuronal damage and colonic dysmotility.Experimental Approach: Oxaliplatin (3 mg·kg⁻¹ per day) was administered in vivo to Balb/c mice intraperitoneally three times a week. The distal colon was collected at day 14 of treatment. Immunohistochemistry was performed in wholemount preparations of submucosal and myenteric ganglia. Neuromuscular transmission was studied by intracellular electrophysiology. Circular muscle tone was studied by force transducers. Colon propulsive activity studied in organ bath experiments and faeces were collected to measure water content.Key Results: Chronic in vivo oxaliplatin treatment resulted in increased formation of reactive oxygen species (O₂ˉ), nitration of proteins, mitochondrial membrane depolarisation resulting in the release of cytochrome c, loss of neurons, increased inducible NOS expression and apoptosis in both the submucosal and myenteric plexuses of the colon. Oxaliplatin treatment enhanced NO‐mediated inhibitory junction potentials and altered the response of circular muscles to the NO donor, sodium nitroprusside. It also reduced the frequency of colonic migrating motor complexes and decreased circular muscle tone, effects reversed by the NO synthase inhibitor, Nω‐Nitro‐L‐arginine.Conclusion and Implications: Our study is the first to provide evidence that oxidative stress is a key player in enteric neuropathy and colonic dysmotility leading to symptoms of chronic constipation observed in oxaliplatin‐treated mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural...

The roles of 5-HT₃ and 5-HT₄ receptors in the modulation of intestinal propulsion by luminal application of 5-HT and augmentation of endogenous 5-HT effects were studied in segments of guinea-pig ileum in vitro. Persistent propulsive contractions evoked by saline distension were examined using a modified Trendelenburg method. When 5-HT (30 nM), fluoxetine (selective serotonin reuptake inhibitor; 1 nM), 2-methyl-5-HT (5-HT₃ receptor agonist; 1 mM), or RS 67506 (5-HT₄ receptor agonist, 1 μM) was infused into the lumen, the pressure needed to initiate persistent propulsive activity fell significantly. A specific 5-HT₄ receptor antagonist, SB 207266 (10 nM in lumen), abolished the effects of 5-HT, fluoxetine, and RS 67506, but not those of 2-methyl-5-HT. Granisetron (5-HT₃ receptor antagonist; 1 μM in lumen) abolished the effect of 5-HT, fluoxetine, RS 67506, and 2-methyl-5-HT. The NK₃ receptor antagonist SR 142801 (100 nM in lumen) blocked the effects of 5-HT, fluoxetine, and 2-methyl-5-HT. SB 207266, granisetron, and SR 142801 had no effect by themselves. Higher concentrations of fluoxetine (100 and 300 nM) and RS 67506 (3 and 10 μM) had no effect on the distension threshold for propulsive contractions. These results indicate that luminal application of exogenous 5-HT, or increased release of endogenous mucosal 5-HT above basal levels, acts to lower the threshold for propulsive contractions in the guinea-pig ileum via activation of 5-HT₃ and 5-HT₄ receptors and the release of tachykinins. The results further indicate that basal release of 5-HT is insufficient to alter the threshold for propulsive motor activity.

Intrinsic sensory neurons (ISNs) of the enteric nervous system respond to stimuli such as muscle tension, muscle length, distortion of the mucosa, and the chemical content in the lumen. ISNs form recurrent networks that probably drive many intestinal motor patterns and reflexes. ISNs express a large number of voltage- and calcium-gated ion channels, some of which are modified by inflammation or repeated physiological stimuli, but how interactions between different ionic currents in ISNs produce both normal and pathological behaviors in the intestine remains unclear. We constructed a model of ISNs including voltage-gated sodium and potassium channels, N-type calcium channels, big conductance calcium-dependent potassium (BK) channels, calcium-dependent nonspecific cation channels (NS_Ca), intermediate conductance calcium-dependent potassium (IK) channels, hyperpolarization-activated cation (I_h) channels, and internal calcium dynamics. The model was based on data from the literature and our electrophysiological studies. The model reproduced responses to short or long depolarizing current pulses and responses to long hyperpolarizing current pulses. Sensitivity analysis showed that I_h, IK, NS_Ca, and BK have the largest influence on the number of action potentials observed during prolonged depolarizations. The model also predicts that changes to the voltage of activation for I_h have a large influence on excitability, but changes to the time constant of activation for I_h have a minor effect. Our model identifies how interactions between different iconic currents influence the excitability of ISNs and highlights an important role for I_h in enteric neuroplasticity resulting from disease.

Segmentation is an important process in nutrient mixing and absorption; however, the mechanisms underlying this motility pattern are poorly understood. Segmentation can be induced by luminal perfusion of fatty acid in guinea pig small intestine in vitro and mimicked by the serotonin (5-HT) reuptake inhibitor fluoxetine (300 nM) and by cholecystokinin (CCK). Serotonergic and CCK-related mechanisms underlying nutrient-induced segmentation were investigated using selective 5-HT and CCK receptor antagonists on isolated segments of small intestine luminally perfused with 1 mM decanoic acid. Motility patterns were analyzed using video imaging and spatiotemporal maps. Segmenting activity mediated by decanoic acid was depressed following luminal application of the 5-HT receptor antagonists granisetron (5-HT₃, 1 μM) and SB-207266 (5-HT₄, 10 nM) and the CCK receptor antagonists devazepide (CCK-1, 300 nM) and L-365260 (CCK-2, 300 nM), but these antagonists did not further depress segmentation when combined. The P2 receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonate (10 μM) had no effect on activity. Serosal application of 5-HT antagonists had little effect on segmentation in the duodenum but reduced activity in the jejunum when granisetron and SB-207266 were applied together. These results reveal that 5-HT₃and 5-HT₄receptors, as well as CCK-1 and CCK-2 receptors, are critical in regulating decanoic acid-induced segmentation. Computational simulation indicated that these data are consistent with decanoic acid activating two pathways in the mucosa that converge within the enteric neural circuitry, while contraction-induced release of 5-HT from the mucosa provides feedback into the neural circuit to set the time course of the overall contractile activity.