Cholera Toxin Induces Sustained Hyperexcitability in Submucosal Secretomotor Neurons in Guinea Pig Jejunum  Rachel M. Gwynne, Melina Ellis, Henrik Sjövall, Joel C. Bornstein  Gastroenterology  Volume 136, Issue 1, Pages 299-308.e4 (January 2009) DOI: 10.1053/j.gastro.2008.09.071 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Schematic diagram of the experimental preparation.The mucosa was removed from half the preparation to reveal the submucosal plexus. The other half consisted of mucosa and submucosal layers. S neurons were impaled in ganglia that were partly covered by the mucosa or had an interganglionic fiber tract running towards the mucosa with no ganglia lying in between (first row). All ganglia impaled further away were analyzed together as a group. Stimulating electrodes were positioned on the mucosa opposite where an interganglionic fiber tract leading from the impaled ganglion disappeared, or on interganglionic fiber tracts leading to the ganglion of choice. The tissue was oriented in the recording bath to avoid the diffusion of substances released from the mucosa towards the recording area. Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 CT increases the number of APs fired and the duration of AP firing during prolonged depolarizations in VIP+ neurons. (A) (I) Confocal image showing 3 S neurons in a submucosal ganglion injected with biocytin during electrophysiological recording and later stained with streptavidin Texas Red. (II) The same field showing both biocytin and VIP immunoreactivity (green). All 3 impaled neurons were VIP+. (B and C) Intracellular recordings from 2 VIP neurons, 1 each from control and CT-treated preparations, respectively. After CT treatment, VIP neurons fired more APs and for significantly longer over a range of depolarizing current pulse amplitudes (50 pA and 250 pA shown here). CT also reduced the voltage threshold for AP firing. Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 CT increases the number of APs fired and the duration of AP firing during prolonged depolarizations in NPY+ neurons. (A) (I) Confocal micrograph showing a neuron injected with biocytin during recording (red). (II) The same field showing the neuron was NPY+ positive (NPY immunoreactivity in green). (B and C) Intracellular recordings from 2 separate NPY neurons showing an increase in number and duration of AP firing after CT treatment. Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 TTX and hexamethonium (Hex) blocked the effects of CT in VIP and NPY neurons. The left panels show the number of APs fired at current pulses between 0.5 and 3.5 nA in control, CT-treated, CT + TTX (1 μmol/L)–treated, and CT+ Hex (200 μmol/L)–treated preparations in VIP neurons (A) and NPY neurons (B). The right panels show the duration of AP firing in the same treatment groups for VIP and NPY neurons. *P < .05 between control and CT-treated preparations; ‡P < .05 between CT and CT + TTX; †P < .05 between CT and CT + Hex preparations. N values are shown. Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Effects of granisetron on CT-induced hyperexcitability in VIP and NPY neurons. (A) Luminal incubation with granisetron (Gran Lum; 1 μmol/L) blocked the CT-induced increased number and duration of AP firing seen in VIP neurons. Serosal application of granisetron (Gran Ser) blocked the increased number of APs fired at high current amplitudes only and had no effect on the increased duration of AP firing in VIP neurons. (B) Granisetron reversed the increased number and duration of AP firing in NPY neurons whether it was added luminally or serosally. (*P < .05 between CT and CT + Gran Lum; †P < .05 between CT and CT + Gran Ser.) Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Effects of blocking NK1 and NK3 receptors on CT-induced hyperexcitability in VIP and NPY neurons. Incubation with the NK1 antagonist SR140333 (100 nmol/L) blocked the effects of CT in both VIP (A) and NPY (B) neurons. The NK3 antagonist SR142801 (100 nmol/L) partially reduced the effects of CT in VIP neurons, reversing the increased number of APs fired but having little effect on the increased duration of AP firing (A). SR142801 reversed the effects of CT in NPY neurons (B). (*P < .05 between CT and CT + SR140333; †P < .05 between CT and CT + SR142801.) Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Anti–CT-B staining was localized to the mucosa in CT-treated preparations. Confocal images of transverse sections (10 μm) of control and CT-treated jejunum after staining with an antiserum against the B subunit of CT. CT-B labeling was localized to the mucosal villi and crypts, but not in the underlying submucosal or myenteric plexuses, or in the muscle layers. CT-B staining was absent in control preparations. Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 CT did not affect the firing of neurons located away from the mucosa. The left panels show the mean numbers of APs fired at current pulses between 50 and 350 pA in control and CT-treated preparations for VIP neurons (A) and NPY neurons (B) impaled more than one row of ganglia away from the mucosa. The right panels show the duration of AP firing in the same neurons. N values are shown. CT did not affect AP firing in neurons located away from the mucosa. Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 CT did not affect the firing properties in calretinin neurons. The left panels show the mean numbers of APs fired at current pulses between 50 and 350 pA in control and CT-treated preparations for (A) all calretinin neurons, (B) calretinin neurons next to the mucosa and (C) calretinin cells in ganglia further than one row away from the mucosa. The right panels show the duration of AP firing in the same neurons. N values are shown. CT did not significantly affect AP firing in calretinin neurons at either location. Gastroenterology 2009 136, 299-308.e4DOI: (10.1053/j.gastro.2008.09.071) Copyright © 2009 AGA Institute Terms and Conditions