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(2008)Conference PaperSerotonin (5-HT) containing enterochromaffin (EC) cells of the intestine detect chemical and mechanical stimuli in the lumen and respond by releasing 5-HT on to afferent nerve terminals. Recent electrochemical studies in healthy mucosa have shown that the real-time release of 5-HT is a dynamic and highly regulated process, but how this might change in disease is unknown. Our aim was to characterize real-time uptake and release of 5-HT in a mouse model of colitis and compare it with ELISA measurements of 5-HT. Real-time electrochemical methods were coupled with an ELISA assay to determine the effect on 5-HT availability of a mouse model of inflammation (5% w/v dextran sodium sulphate (DSS) induced colitis). Peak and steady state (SS) 5-HT concentrations (calculated from the oxidation current at +400 mV; amperometry mode) were measured with our without the serotonin reuptake transporter blocker fluoxetine (1 lM) in control and DSS-treated mice. Paired and unpaired data were compared with a one way ANOVA (P < 0.05). In mouse colon, SS release of 5-HT was 1.9 ± 0.6 lM (n = 9) and compression-evoked release was 7.1 ± 2.5 lM (n = 9). In DSS treated mice, the release of 5-HT was significantly increased (SS: 3.4 ± 0.6 lM; peak: 14.7 ± 3.0 lM; n = 11). In control mice, fluoxetine significantly increased peak (9.9 ± lM) but not SS release (2.6 ± 0.4 lM), while in DSS mice both were significantly increased (SS: 7.3 ± 1.2 lM; peak: 23.4 ± 4.1 lM). The effects of fluoxetine in DSS mice were greater than in control. ELISA assays supported these data, showing an increase in 5-HT release detected from inflamed colon (n = 5) compared to control (n = 5) in unstimulated or mechanically stimulated preparations and with or without fluoxetine. The release and uptake of 5-HT from the EC cells of the mouse colon are increased during DSS colitis. Our electrochemical data show that both peak and steady state levels are increased and these changes are mirrored by the ELISA data. In addition, the localised 5-HT concentrations at the site of release, measured using amperometry, are significantly higher than those reported using ELISA techniques. Overall, these data show that during colitis, 5-HT availability will be increased. These raised concentrations may substantially alter the activation or desensitisation of serotonin receptors on afferent nerve terminals.
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(2007)Conference PaperBackground/Aims: Enterochromaffin (EC) cells are sensors in the intestinal lumen that detect chemical or mechanical stimuli and respond with the release of serotonin (5-HT). 5-HT can activate local reflexes but whether local activation of enteric neural circuits also evoke 5-HT release is unclear. Methods: Recordings were made from full-thickness preparations of guinea pig ileum using electrochemical techniques with carbon fibre electrodes placed in the mucosa to measure local concentrations of 5-HT. The tension in the circular muscle (CM) was recorded with a force transducer. Amplitude and time course of local 5-HT release events and muscle contractions were measured and compared using paired Student’s t-test with Bonferroni’s correction for multiple comparisons. Results: Stretch of the CM caused 5-HT release and reflex contraction of the CM. Focal electrical stimulation of the intestine near to the carbon fibre electrode evoked 5-HT release and caused a local contraction in the ring of CM where the recording site was located. Paralysis of the smooth muscle with papaverine (100 mm; n55), sodium nitroprusside (100 mm; n55) or isoproterenol (1 mm; n55) significantly reduced (o25% of control) the stretch-evoked release of 5-HT. Similarly, isoproterenol (1 mm; n54) or sodium nitroprusside (100 mm; n53), abolished (o10% of control) the electrically evoked release of 5-HT. Atropine (1 mm; n53), which would be expected to block muscarinic input to the EC cell, did not reduce stretch-evoked 5-HT release. Conclusion: The present study provides direct evidence that activated enteric nerves are not responsible for the 5-HT release seen during local reflexes. There was little residual 5-HT release in response to stretch or electrical stimulation of the nerves in paralysed preparations and atropine did not reduce reflex-evoked 5-HT release. Together, these data suggest that mechnical, and not neural, stimuli provide an important excitatory input to the EC cells.
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(2003)Conference PaperThe pattern of input to intestinal sensory neurons shapes their output and is, thus, fundamental to an understanding of the initiation and propagation of intestinal reflexes. Recent data suggests that this input consists largely of bursts of action potentials (APs). The origin of these bursts in 45 myenteric sensory neurons from guinea pig ileum was investigated. All neurons were near intact mucosa and intracellular electrophysiological recording techniques were used during selective electrical stimulation of the mucosal epithelium and of the cell body. Three types of bursts were seen: following an antidromic AP evoked by electrical stimulation of the mucosa; following an AP evoked at the soma; and spontaneously occurring. All bursts had an average intra-burst frequency of between 40-50 Hz and tended to occur in the same neurons. All APs in a burst persisted during somatic hyperpolarization and many had an inflection on their rising phase. They also persisted in low calcium solutions and a variety of ligand-gated ion channel receptor antagonists. TTX (300nM) abolished all bursts while TTX (30nM), tropisetron (30microM) or a high calcium, high magnesium solution selectively blocked the later APs in a burst. Results from collision experiments suggest that both the electrically and somatically evoked bursts originate near the sensory nerve terminals in the mucosa. These bursts of APs are widespread and have many characteristics in common suggesting similar mechanisms underlie generation of each. These data suggest that positive feedback between sensory nerve terminals and epithelial elements may be important in normal intestinal function.
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(2003)Conference PaperThe pattern of input to intestinal sensory neurons shapes their output and is, thus, fundamental to an understanding of the initiation and propagation of intestinal reflexes. Recent data suggests that this input consists largely of bursts of action potentials (APs). We investigated the origin of these bursts in 60 ileal sensory neurons taken from guinea pigs that were stunned by a blow to the head and killed by severing the carotid arteries. All myenteric neurons were near intact mucosa and intracellular electrophysiological recording techniques were used during selective electrical stimulation of the mucosal epithelium and of the cell body. Three types of bursts were seen: following an antidromic AP evoked by electrical stimulation of the mucosa (66% of neurons); following an AP evoked at the soma (30%); and spontaneously occurring (38%). All bursts had an average intra-burst frequency of 50 Hz and all tended to occur in the same population of neurons. All APs in a burst persisted during somatic hyperpolarization and many had an inflection on their rising phase. They also persisted in low calcium solutions and a variety of ligand-gated ion channel receptor antagonists. TTX (300 nM) abolished all bursts while TTX (30 nM), tropisetron (30 microM) or a high calcium, high magnesium solution selectively blocked the later APs in a burst. Results from collision experiments suggest that both the electrically and somatically evoked bursts originate near the sensory nerve terminals in the mucosa. The characteristics of the bursts of APs are common, suggesting a common mechanism underlies each. This involves positive feedback between nerve terminals and epithelial elements and may be important in differentiating or integrating sensory modalities.
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(1998)Conference PaperThere are similarities between the mechanisms of sensory transduction by chemoreceptors in the gastrointestinal (GI) tract and by those in the oral cavity. Dissociated rat taste receptor cells have been shown to contain vesicular 5-HT and are excited by exogenous application of 5-HT. Likewise, in the GI tract, enterochromaffin (EC) cells are well known to contain 5-HT and are excited by its exogenous application. Both the taste receptor cell and the EC cell apparently make reciprocal synaptic connections with their extrinsic and (in the case of the gut) intrinsic afferent innervation. However, while the taste receptor cell is known to participate in gustation, the EC cell's role in the initiation of enteric reflexes remains unclear. Thus it may be that common transduction mechanisms are utilized in both systems and it is in this sense that the gut may be said to 'taste' the contents of the lumen. These studies were designed to investigate the sensory transduction mechanisms underlying responses of myenteric sensory neurons to chemical stimulation of the mucosa and with particular attention to the possible interactions between the EC cell and the surrounding afferent innervation. Intracellular electrophysiological recordings were taken from chemosensory myenteric neurons which had AH type electrophysiological characteristics and Dogiel type II morphological characteristics. Short segments of guinea pig ileum were dissected to reveal the myenteric plexus over one half the circumference of the preparation. Focal stimulating electrodes and fine glass pipettes containing chemical substances were used to stimulate the mucosa in the intact half of the preparation. Movement of the smooth muscle was reduced with the addition of scopolamine and nicardipine. In general, stimulation of the mucosa generated bursts of one or more action potentials (APs) recorded at the cell soma. Puffs of 5-HT (3 - 30 M) or 2-methyl 5-HT (0.1 to 1 mM - a 5-HT3 agonist) applied locally to the mucosa were effective in eliciting bursts of APs while puffs of -methyl 5-HT (0.1 to 1 mM - a 5-HT2 agonist) or 5-methoxytryptamine (1 to 10 M - a general 5-HT agonist) were not. Single, electrical stimuli applied to the mucosa elicited a single antidromic AP and a later burst of APs. Spontaneous bursts of APs were also recorded in some cells which were indistinguishable from the electrically evoked late burst of APs. In one instance, initiation of an antidromic AP by electrical stimulation of the mucosa or a single AP by somatic current injection elicited a late AP which appeared to be the result of synaptic interaction at or near the nerve terminal in the mucosa. In cells with spontaneous bursts of APs or where 5-HT elicited a burst of APs, application of tropisetron (10 M - 5-HT3/4 antagonist) reduced the occurrence of bursts and/or the number of APs in a burst. The electrically evoked late burst of APs was only blocked by higher concentrations of tropisetron (30 M) and was not desensitized during repeated applications of 5-HT to the mucosa. The early, antidromic AP was never blocked. In preliminary experiments, granisetron (10 M - 5-HT3 antagonist) reduced the number of APs in response to 5-HT but not to electrical stimulation. These results are consistent with the idea that 5-HT participates in chemosensory transduction, but also suggest that other substances may play a similar role. In addition, anecdotal evidence suggest that the mucosa and its afferent innervation can interact in a positive, reciprocal fashion.
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(2008)Conference Paper
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(2003)Conference Paper