Volume 137, Issue 4, Pages 1425-1434 (October 2009) Activation of Human Enteric Neurons by Supernatants of Colonic Biopsy Specimens From Patients With Irritable Bowel Syndrome  Sabine Buhner, Qin Li, Sheila Vignali, Giovanni Barbara, Roberto De Giorgio, Vincenzo Stanghellini, Cesare Cremon, Florian Zeller, Rupert Langer, Hannelore Daniel, Klaus Michel, Michael Schemann  Gastroenterology  Volume 137, Issue 4, Pages 1425-1434 (October 2009) DOI: 10.1053/j.gastro.2009.07.005 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 IBS but not HC supernatants evoked action potential discharge in human submucosal plexus neurons. (A) Di-8-ANEPPS labeled ganglion revealing outline of dye labeled individual neurons. Signals from 2 of the neurons are shown in traces A-1 and A-2. Application of supernatants was 200 ms (indicated by bars below the traces). Neuron A-1 did not respond to application of HC supernatant. In contrast, D-IBS 23 supernatant evoked discharge of action potentials. Neuron A-2 did not respond to HC or IBS supernatants. Panel B shows representative traces to illustrate the similarity of the responses to D-IBS (middle trace) and C-IBS supernatants (bottom trace). The number of action potentials (AP) in the particular trace is given in the right upper corner. Gastroenterology 2009 137, 1425-1434DOI: (10.1053/j.gastro.2009.07.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Compared with HC, each of the IBS supernatants significantly increased spike discharge in 40%–100% of the neurons per ganglion. Data for HC supernatants were pooled. Proportions of activated neurons are given as median percentage (25th and 75th percentiles); (T/G/N): number of tissues/ganglia/neurons; *P < .05 compared with HC. Gastroenterology 2009 137, 1425-1434DOI: (10.1053/j.gastro.2009.07.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Serotonin receptor (5-HT3), histamine receptor (H1, H2, H3) blockade, and protease inhibition inhibited IBS supernatant-evoked spike discharge. Panels A–C show neuron responses after control application IBS supernatant, reapplication with blockers, and after washout of blockers. (A) IBS supernatant-evoked spike discharge is reduced by the 5-HT3 receptor antagonist cilansetron and recovers after 40-minute washout. (B) IBS supernatant evoked spike discharge is reduced by the combined application of the H1, H2, and H3 antagonists ranitidine, pyrilamine, and clobenpropit, respectively. (C) Preincubation of the IBS supernatant with the protease inhibitor FUT-175 blocked the spike discharge. A second application of the IBS supernatant still evoked a response. The number of action potentials (AP) in the particular trace is given in the right upper corner. Gastroenterology 2009 137, 1425-1434DOI: (10.1053/j.gastro.2009.07.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 (A) Tryptase and histamine but not serotonin concentrations were significantly higher in IBS supernatants compared with supernatants from HC; crosses next to dot plots indicate medians and interquartile ranges (25th and 75th percentiles). (B) Tryptase, histamine, and serotonin concentrations in the supernatants significantly correlated with the supernatant-evoked action potential discharges. Gastroenterology 2009 137, 1425-1434DOI: (10.1053/j.gastro.2009.07.005) Copyright © 2009 AGA Institute Terms and Conditions