Volume 132, Issue 5, Pages 1912-1924 (May 2007) An Enteric Occult Reflex Underlies Accommodation and Slow Transit in the Distal Large Bowel  Eamonn J. Dickson, Nick J. Spencer, Grant W. Hennig, Peter O. Bayguinov, Jim Ren, Dante J. Heredia, Terence K. Smith  Gastroenterology  Volume 132, Issue 5, Pages 1912-1924 (May 2007) DOI: 10.1053/j.gastro.2007.02.047 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Effects of circumferential and longitudinal stretch. (A) Circumferential stretch applied to the distal colon activated ongoing peristaltic reflex activity consisting of oral excitatory junction potentials (EJPs) that were coordinated with anal inhibitory junction potentials (IJPs) in the circular muscle. Transmural nerve stimulation (transmural stimulating single pulse 0.5 ms, 30 V) applied to the middle of the preparation evoked a robust oral EJP and anal IJP. (B) When the same preparation was stretched only in the longitudinal axis, only low-amplitude neural activity was observed and gave only a small response to TS. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Effect of blocking nitric oxide production. (A) Control: ongoing oral EJPs and anal IJPs in another circumferentially stretched preparation. Black arrow indicates onset of TS (single pulse 0.5 ms, 30 V). (B) Ongoing peristaltic reflex activity evoked by circumferential stretch and oral EJPs and anal IJPs evoked and TS were almost abolished by 60% longitudinal stretch. (C) L-NA (100 μmol/L) added to the middle chamber restored all ongoing and evoked activity. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Correlation between EJPs and IJPs. (A) Correlation coefficient (R2; n = 6) for temporal coordination between ongoing oral EJPs and anal IJPs evoked by circumferential stretch (1) for control-circumferential stretch only; (2) following the addition of 60% longitudinal stretch, and (3) following removal of longitudinal stretch but not circumferential stretch. (B) Amplitude of oral EJPs plotted against the amplitude of anal IJPs (1) during circumferential stretch, (2) following the addition of 60% longitudinal stretch, and (3) after removing only longitudinal stretch. Note that the noise bubble shows that EJPs and IJPs <5 mV were uncoordinated. (C) Correlation coefficient (R2) for temporal coordination between ongoing oral EJPs and anal IJPs evoked by circumferential stretch (1) for circumferential stretch only, (2) following 25% longitudinal stretch, (3) following the addition of 60% longitudinal stretch, and (4) following 60% longitudinal stretch and L-NA (100 μmol/L) in middle chamber. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Effects of transmural nerve stimulation and removal of longitudinal muscle. (A) Evoked oral EJP and (B) anal IJP amplitude plotted against different strengths of TS (single pulses 0.5 ms, 0–75 V; n = 5) (1) in a circumferential stretched preparation (solid black line with diamond); (2) following the addition of 60% longitudinal stretch (dashed line with square); and (3) following the further addition of L-NA (100 μmol/L) (dashed line with triangle) in the middle chamber. (C) Following removal of longitudinal muscle, circumferential stretch evoked normal ongoing coordinated oral EJP and anal IJP activity. (D) Despite removal of the longitudinal muscle, stretching the same segment of distal colon as used in Panel C to 60% beyond the slack length still depressed activity generated by circumferential strength. (E) Following the removal of longitudinal stretch, the activity evoked by circumferential stretch returned. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Ca2+ imaging of myenteric neurons following blockade of synaptic neurotransmission. A segment of colon, with the longitudinal muscle removed, was pinned in both axes. Hexamethonium (100 μmol/L), ondansetron (1 μmol/L), and PPADs (30 μmol/L) were added to the Krebs’ solution to block fast synaptic transmission. (A) Average Ca2+ fluorescence in myenteric neurons. (B) An overlay of nNOS immunoreactivy (red) superimposed on averaged Ca2+ fluorescence. (C) nNOS immunoreactivy (red) overlaid with average Ca2+ fluorescence in L-NA (100 μmol/L). (D) Before the addition of L-NA, only 1 neuron in this ganglia exhibited ongoing activity (see arrowhead 1 in A–C). This neuron was nNOS +ve and anally projecting (see arrowhead 1 in A and B). (E) L-NA (100 μmol/L) did not effect the activity of the NOS +ve neuron (black trace) but revealed activity in other neurons (boxes 2–5; C and E) that were NOS −ve. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 NO-mediated synaptic events. In the presence of guanethidine (0.5 μmol/L) and nifedipine (1 μmol/L), recordings were made from myenteric neurons in a circumferentially stretched CMMP preparation that were situated approximately 10 mm anal of transmural stimulating (TS) electrodes. (A) IPSP evoked in S neuron exhibiting spontaneous action potentials (0.5-ms duration, 5 pulses at 10 Hz, 30 V). (B) A similar stimulus evoked FEPSPs in a neuron with ongoing FEPSPs. (C) IPSP evoked in another neuron in the presence of quanethidine (0.5 μmol/L). This neuron exhibited spontaneous action potentials, FEPSPs, and proximal process potentials. (D) Following the further addition of L-NA (100 μmol/L), the IPSP in the same neuron was blocked. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Effects of colonic elongation on pellet propulsion. (A) As shown in the spatiotemporal map, an epoxy-coated pellet was allowed to propel spontaneously down the distal colon. Once propulsion was initiated, the pellet propelled at a constant velocity down the colon. (B) In another run, once the pellet started to move at a constant velocity, colonic elongation was applied to the oral flap. Elongation of the flap to approximately twice its resting length almost stopped pellet propulsion. Following removal of the stimulus, the flap gradually relaxed back to its resting length, and the pellet then continued to move down the colon at its prestretched velocity. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Colonic filling leads to elongation. (A) Organ bath for filling a segment of colon with fluid and video imaging its movements. A tension transducer was attached to the circular muscle at the oral end of the colon. (B) Graph showing diameter, length, and tension changes in the circular muscle during increases in intraluminal volume (n = 5). L-NA (100 μmol/L) increased tone during colonic elongation. (C) An example of colonic motor activity during filling expressed as a spatiotemporal map. Once the colon reached its near maximal level of circumferential distension, it elongated with a consequent drop in contractile activity in both the longitudinal and the circular muscle. Withdrawal of injected fluid increased contractions of colon, which gradually assumed its resting length. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions

Figure 9 Proposed neural circuit underlying colonic elongation. (A) Schematic shows that circumferential stretch activates mechanosensitive ascending excitatory (AEPs) and descending inhibitory nerve pathways (DIPs) underlying peristalsis. AEPs and DIPs, which reinforce one another, activate excitatory motor neurons (EMNs) orally and inhibitory motor neurons (IMNs) anally to both the longitudinal and circular muscle layers. Activation of EMNs produces an excitatory junction potential (EJP), and activation of IMNs produces an inhibitory junction potential (IJP) in the circular muscle, at the oral and anal ends of the segment, respectively. During circumferential stretch, descending NOS neurons are quiescent. (B) Following colonic elongation, descending NOS +ve interneurons are activated to release NO, which likely generates IPSPs in mechanosensitive interneurons in AEPs and DIPs and a consequent withdrawal of activity from both EMNs and IMNs. Gastroenterology 2007 132, 1912-1924DOI: (10.1053/j.gastro.2007.02.047) Copyright © 2007 AGA Institute Terms and Conditions