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Previous studies have indicated that neither neuronal nor mucosal 5-hydroxytryptamine (5-HT) are important for colonic migrating motor complexes (CMMCs) or faecal pellet propulsion. Therefore, tryptophan hydroxylase 1 knockout (TPH1KO) mice were used to examine the role of mucosal 5-HT in generating CMMCs and faecal pellet propulsion, as TPH1 is the regulatory enzyme necessary for the synthesis of 5-HT in enterochromaffin cells in the mucosa. Control mice generated a robust CMMC when the mucosa was mechanically stimulated, which was blocked by ondansetron (5-HT3 antagonist), and could propagate faecal pellets that did not significantly distend the bowel, suggesting that they were propelled by mucosal reflexes in the absence of stretch reflexes. TPH1KO mice exhibited no mucosal reflexes, reduced responses to intraluminal distension and propelled only larger faecal pellets, suggesting that they relied upon stretch reflexes alone. In control mice, CMMCs, which can propel a faecal pellet, propagated in an oral to anal direction, whereas, in TPH1KO mice, they rarely propagated. Both the propagation and amplitude of CMMCs were reduced by ondansetron in control mice, whereas this drug did not affect CMMCs in TPH1KO mice. This suggests that 5-HT release from the mucosa and stretch reflexes are important for normal colonic propulsion. Abstract Although there is general agreement that mucosal 5-hydroxytryptamine (5-HT) can initiate peristaltic reflexes in the colon, recent studies have differed as to whether or not the role of mucosal 5-HT is critical. We therefore tested the hypothesis that the secretion of 5-HT from mucosal enterochromaffin (EC) cells is essential for the manifestation of murine colonic peristaltic reflexes. To do so, we analysed the mechanisms underlying faecal pellet propulsion in isolated colons of mice lacking tryptophan hydroxylase 1 (Tph1−/− mice), which is the rate-limiting enzyme in the biosynthesis of mucosal but not neuronal 5-HT. We used video analysis of faecal pellet propulsion, tension transducers to record colonic migrating motor complexes (CMMCs) and intracellular microelectrodes to record circular muscle activity occurring spontaneously or following intraluminal distension. When compared with control (Tph1+/+) mice, Tph1−/− animals exhibited: (1) an elongated colon; (2) larger faecal pellets; (3) orthograde propulsion followed by retropulsion (not observed in Tph1+/+ colon); (4) slower in vitro propulsion of larger faecal pellets (28% of Tph1+/+); (5) CMMCs that infrequently propagated in an oral to anal direction because of impaired descending inhibition; (6) reduced CMMCs and inhibitory responses to intraluminal balloon distension; (7) an absence of reflex activity in response to mucosal stimulation. In addition, (8) thin pellets that propagated along the control colon failed to do so in Tph1−/− colon; and (9) the 5-HT3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1+/+ mice, failed to alter CMMCs in Tph1−/− animals. Our observations suggest that mucosal 5-HT is essential for reflexes driven by mucosal stimulation and is also important for normal propagation of CMMCs and propulsion of pellets in the isolated colon. 5-Hydroxytryptamine (5-HT, serotonin) is highly abundant in the gut (∼95% of the 5-HT in the body) and participates in neurocrine, paracrine and endocrine signalling (Gershon, 2012, 2013). Enteric sources of 5-HT include mucosal enterochromaffin (EC) cells, mast cells in rats and mice, and myenteric serotonergic neurons, which are descending interneurons that also project to the submucous plexus (Sang et al. 1997; Lomax Li et al. 2011; Faure et al. 2010). Secretion of 5-HT from EC cells can activate intrinsic primary afferent neurons (IPANs) that initiate motility and secretomotor reflexes (Bülbring Pan Heredia et al. 2009; Bayguinov et al. 2010). Colonic descending serotonergic interneurons, which activate inhibitory motor neurons, produce tonic inhibition of the circular muscle and promote secretion (Dickson et al. 2010b; Heredia et al. 2012; Okamoto et al. 2012). 5-HT3 receptor antagonists are effective against irritable bowel syndrome with diarrhea (Mangel Heredia et al. to peristaltic are not to on the distension of the gut and stretch can also do so Bayguinov et al. 2010). to of the mucosa and 5-HT were to activate peristaltic reflexes only stretch mucosal stimulation also peristaltic reflexes that are to by stretch Pan Bayguinov et al. with interneurons in the myenteric plexus et al. et al. a EC cells and stretch receptors to reflexes. was to the hypothesis that mucosal secretion of 5-HT is necessary for the of peristaltic reflexes and normal faecal pellet in the isolated murine colon. To do we compared the reflex responses and faecal pellet propagation in isolated of colon from control (Tph1+/+) mice with from mice in which tryptophan hydroxylase 1 was TPH1 is the rate-limiting enzyme for 5-HT biosynthesis in mucosal EC and mast cells, and so Tph1−/− mice mucosal 5-HT et al. et al. Li et al. 2011; et al. 2012). is the rate-limiting enzyme for 5-HT biosynthesis in and myenteric serotonergic neurons, neuronal 5-HT is in Tph1−/− mice. Tph1−/− mice and (Tph1+/+) were on a et al. were to the of they were in the the of of mice were on a with and were by a of followed by was and the colon colon and was were in with of for the and of animals and by the the of and with in the as by pellets from of mice in were for 1 and their was to the faecal pellets were from the isolated colon with a with were when and in an were with a on a faecal pellets colon were of a colon isolated colon, faecal pellets, was in an with in the oral and in the anal were to the of the colon for video pellet propulsion colon was in the were in the oral to or faecal pellets to In faecal pellets of were were by and to pellet was the oral and the was with a video to a and analysed with this were generated by a and in a descending to were so that the could from to This of the the pellet to to not the oral and anal of the colon are the to as the and of the in is the of a pellet in the bowel the by a of from to et al. 2012). 1 was and to an faecal pellet was the and to the of the the in with the pellet located in the of the colon tension transducers were by to the colonic in to the tension of the circular the was to the colon by a of and the tension was to so as to not stretch the colon tension transducers were from the oral of the colon the colonic and from the anal were for CMMCs were observed distension balloon was for the pellet when the of distension were balloon was the and transducers with was to the and used to the balloon when the of a faecal were by a by of a and with and amplitude of complexes were reflexes of mucosa was on the and for as a to the mucosa or by on the mucosa with a Heredia et al. of the colon of the colon tension was by tension from the a of the colon and tension was to et al. 2010). colon was along the and with the mucosa against the of the balloon was the and in the of the colon for distension from the muscle cells were by a the muscle the underlying circular as et al. et al. of CMMCs was the amplitude of and with the of were a following the drug were with an and a with for were of were or or and was as to the of animals from which colons were are as In the and and ondansetron were from with mice were in to their of the Tph1−/− colon was significantly that of and faecal pellets were larger in Tph1−/− in Tph1+/+ mice the of were the were significantly in Tph1−/− mice and pellets from Tph1−/− mice were and pellets, in Tph1+/+ mice Colonic and faecal pellets of and colons of Tph1+/+ and Tph1−/− mice. of faecal pellets from Tph1+/+ and Tph1−/− mice. colonic in and colons from Tph1+/+ and Tph1−/− mice. of faecal pellet from Tph1+/+ and Tph1−/− mice. of faecal pellet and in Tph1+/+ and Tph1−/− mice. of faecal pellets was in Tph1−/− pellets in Tph1+/+ pellets animals the faecal was in Tph1−/− in Tph1+/+ mice. faecal pellet in faecal pellet was in Tph1−/− and in Tph1+/+ mice and This suggests that Tph1−/− mice larger of did Tph1+/+ mice. faecal pellet which was to the larger faecal pellets in the Tph1−/− was used to the propulsion of pellets the isolated colon. pellets propagated along the of the isolated colon, but did so in Tph1−/− in Tph1+/+ This in pellet was observed the colon and In to the slower pellet in Tph1−/− colons was observed in Tph1−/− but not in Tph1+/+ colon. propulsion with propulsion that was to faecal pellet pellet propulsion was in Tph1+/+ colons and in the colon of an faecal pellet in the Tph1−/− with the pellet to orthograde and propulsion in the is from to whereas on the and on the pellet in an oral to anal direction, not of the that orthograde propulsion is indicated by a a and propulsion is indicated by a a and of pellet propulsion in Tph1−/− and Tph1+/+ mice. of faecal in a Tph1−/− faecal pellet the colon, colon and of Tph1+/+ and Tph1−/− mice. When the colon of faecal pellets was from the and in an the for was in Tph1−/− in Tph1+/+ of the faecal pellets, which activate only stretch reflexes, have to propagate an isolated colon et al. 2011; et al. we whether mucosal reflexes in the absence of could propagate faecal faecal pellets propagated isolated Tph1+/+ of pellet propagation was upon the pellet faecal pellets but not propagated of because thin pellets failed to on the mucosa to release 5-HT to initiate a peristaltic reflex Heredia et al. of receptors and reflexes tension of colonic migrating motor complexes (CMMCs) occurring in a Tph1−/− colon. blocked CMMC activity in a Tph1−/− colon. CMMCs occurring in a Tph1−/− colon. of colonic activate the reduced CMMCs in the Tph1−/− of the isolated colon a inhibitory which the amplitude of CMMCs in Tph1+/+ colon et al. 2010). of were also observed in Tph1−/− colon and because the colons of mice are an inhibitory to to faecal pellet in 2012). Colonic migrating motor complexes (CMMCs) by of reflexes in Tph1−/− mice CMMC by balloon distension in Tph1+/+ and Tph1−/− colon. CMMCs by balloon distension in Tph1+/+ and Tph1−/− mice as a of CMMC in the Tph1+/+ colon. oral to anal CMMC propagation in Tph1+/+ but not in Tph1−/− mice. mucosal stimulation a CMMC in Tph1+/+ colon, but not in Tph1−/− colon. CMMC by oral and anal mucosal stimulation in Tph1+/+ and Tph1−/− colon. CMMC can in response to stretch or stimulation of the mucosa with an the mucosal response is reduced or blocked by 5-HT3 antagonists et al. 2009; Bayguinov et al. 2010). of an intraluminal which to activate stretch and mucosal reflexes, the and tension a robust CMMC in Tph1+/+ colon, but a significantly reduced response in colons of TPH1KO mice and distension of the Tph1−/− colon responses that were of the of observed in Tph1+/+ colon of balloon were from the circular muscle anal of the of distension distension a of in the circular muscle of distension a response ondansetron in to balloon distension in Tph1−/− colon. to balloon distension in Tph1−/− colon ondansetron amplitude and of the by balloon distension in Tph1+/+ and Tph1−/− colon, and ondansetron and following the of that ondansetron and also reduced the of the inhibitory response in Tph1−/− colon. the colonic mucosa with an to the mucosa the oral of the colon or to the mucosa the anal also a robust CMMC in Tph1+/+ colon; CMMCs were blocked by ondansetron stimulation of the Tph1−/− colon, failed to CMMCs and circular muscle of the colon to tonic inhibition; inhibitory CMMCs et al. Heredia et al. 2012; et al. interneurons with inhibitory motor neurons that the circular muscle et al. 2012; et al. In the we observed in the circular muscle of Tph1+/+ and Tph1−/− mice and in Tph1−/− colon was significantly that in Tph1+/+ colon In Tph1−/− an amplitude of which was that of in Tph1+/+ colons of the circular muscle in Tph1+/+ and Tph1−/− mice were not significantly In Tph1−/− and Tph1+/+ colon, oral intraluminal balloon distension in a of of the circular muscle which was followed by a CMMC and Both the and the were significantly in Tph1−/− in Tph1+/+ mice. amplitude of the was in Tph1−/− and Tph1+/+ mice significantly reduced the and the in Tph1−/− and Tph1+/+ colon Although ondansetron reduced the amplitude of the in Tph1−/− colon, on this in colons of Tph1+/+ mice was not of reduced the amplitude and of the in Tph1−/− and Tph1+/+ mice the nor inhibition of TPH1 to alter murine gut bowel propulsion or of of the et al. Li et al. observations have to the that mucosal 5-HT not essential for gastrointestinal In of this we that the faecal of Tph1−/− mice was to or that of control mice. In faecal pellet propulsion in the isolated colon was only of that of Tph1+/+ in with the Tph1+/+ colon, the Tph1−/− (1) was (2) larger faecal pellets; (3) a (4) propulsion of faecal pellets; (5) only rarely propagated CMMCs in an oral to anal direction, because of a of descending inhibition; (6) exhibited CMMCs in the absence of mucosal (7) no mucosal (8) exhibited reduced CMMCs and inhibitory responses to intraluminal distension; and (9) CMMCs that were to ondansetron (5-HT3 in vitro colonic motility is in Tph1−/− mice, which mucosal 5-HT. elongated colon, faecal pellets and of mucosal reflexes suggest that mice of constipation Heredia et al. 2012). normal in is a of in mice, which have et al. and and et al. In of the that mucosal 5-HT is important for normal colonic propulsion of faecal pellets, we that thin pellets, which did not significantly distend the colon, propagated the isolated Tph1+/+ colon, but not the Tph1−/− colon, that mucosal reflexes stretch reflexes, are of faecal and in studies of isolated colons from and that peristaltic reflex could not from from which the mucosal and are essential for the of the peristaltic reflex and for the propulsion of in the and reflexes in the normal propagation of faecal pellets in the colon in reflexes to in with mucosal reflexes, stretch reflexes et al. the of reflex to a that reflexes to to of reflex as a of reduced muscle which activity in et al. et al. or mucosal or the peristaltic that the of gastrointestinal in Tph1−/− mice that mucosal which is in no to peristaltic reflexes in the colon. the observations that mucosal 5-HT a to normal peristaltic reflexes in the murine colon. studies also suggest that 5-HT is the not the paracrine from EC cells by stimulation of the as et al. 2009; Bayguinov et al. 2010). faecal pellet 5-HT from the which in activate descending inhibitory et al. 2012). inhibition is generated by the robust of serotonergic neurons which activate inhibitory motor neurons 5-HT3 receptors et al. 2012; et al. CMMCs have an of et al. which is slower along the myenteric plexus et al. the of the inhibitory and muscle responses are and the of the muscle et al. 2010). not for the inhibitory response in the which the of the CMMCs to along the colon et al. as they do in the of 5-HT is to a primary in this the (Dickson et al. as by recent studies et al. 2013). oral to anal of CMMCs in Tph1−/− mice that are not the inhibition that the CMMC because they are not by mucosal 5-HT et al. 2009; Bayguinov et al. et al. suggests that there is not for of the in of a faecal pellet, to a CMMC that and a faecal This could the by a faecal pellet in Tph1−/− mice in with the a pellet in the control colon. CMMCs were observed in Tph1−/− mice the absence of mucosal as they are in mice as a of a in which an of mucosal reflexes because the release of 5-HT et al. 2012). observations suggest that the colon to a of mucosal 5-HT by the of as in mice Bayguinov et al. et al. CMMCs are to the of 5-HT3 receptors with ondansetron et al. Heredia et al. which also the (Dickson et al. In with CMMCs, by are to ondansetron (Dickson et al. suggesting that the drug not CMMC CMMCs and pellet propulsion have to in mice and with to 5-HT et al. et al. 2013). ondansetron et al. and the 5-HT4 antagonist et al. have to CMMCs and faecal pellet propulsion in mice and when the mucosa is of CMMCs in gut used to a hypothesis that neither EC nor neuronal 5-HT a role in or is an of the and only with the of 5-HT from to Heredia et al. 2009; Bayguinov et al. et al. and by interneurons in as by descending serotonergic neurons et al. as for the et al. & to intraluminal including CMMCs and anal of in Tph1−/− colon were robust in isolated Tph1+/+ colon, suggesting that responses to intraluminal distension are on the secretion of 5-HT from EC serotonergic neurons, on the 5-HT3 receptors by inhibitory motor neurons, also to the responses to distension in Tph1−/− and Tph1+/+ mice. propulsion was followed by faecal pellet propulsion in the isolated Tph1−/− colon. a was not observed in Tph1+/+ mice, there is a in the of CMMC in Tph1−/− colon. propulsion is by CMMC that propagate in the anal to oral CMMCs in the Tph1+/+ colon propagate in an oral to anal direction of the whereas of CMMCs in the Tph1−/− colon propagate to larger faecal pellets of Tph1−/− mice is with the that faecal in in the colon of Tph1−/− in that of Tph1+/+ mice. This is with the that pellets are propelled in isolated Tph1−/− in Tph1+/+ colon. in the of the colon and the propulsion of pellets in vitro is in the Tph1−/− colon and of the isolated colon. of propulsion, were also to in also to the of the faecal pellets of Tph1−/− mice. faecal pellets, elongated colons and propagation of CMMCs in Tph1−/− mice are that constipation were to in Tph1−/− mice et al. 2012). the absence of peristaltic reflexes in Tph1−/− mice pellet to to they are to reflexes in for propulsion to of the bowel are not when in are only the is which is an of are of for a to the bowel, from the or from the are and do not reflexes the gut to of as mucosal stimulation or distension. in vitro in the gut to but they do not whether or are in of motility therefore in and in vitro suggests that mucosal 5-HT a to reflex activity of the In reflexes can of mucosal 5-HT. is that this for of the in the the role of mucosal 5-HT. is also in a propulsion can in the gut as in a Tph1−/− the bowel is to peristaltic reflexes Tph1−/− animals and their gastrointestinal is in vitro that their bowel is to of reflex is important to to in the manifestation of the of reflex activity that the in vitro studies is in Tph1−/− mice. is that the of Tph1−/− mice to from the is with that of Tph1+/+ mice et al. used in studies a of which is to the of faecal pellets observed in Tph1−/− mice. of from the of Tph1−/− mice in normal mucosal reflexes are because stretch reflexes are necessary to examine the of of to Tph1+/+ and Tph1−/− mice. the mucosal 5-HT of the in vitro peristaltic reflex in the colon and the mucosal 5-HT of the in gastrointestinal suggests that to the peristaltic reflex by and in the is not essential for gastrointestinal motility have in the that to the peristaltic reflex is propulsion, which not in the bowel is or the mucosa is by a as or of motility to propulsion when mucosal reflex activity is that Tph1−/− mice CMMCs in as a of distension of the bowel by the faecal pellets in the colon, Tph1−/− gut can propel to are with a or in a peristaltic as propulsion or so, the or of a Tph1−/− which to a Our in vitro studies of isolated colon suggest that there is in the of colonic motility in Tph1−/− mice which is not in the of the gastrointestinal in the murine gut in This a of the of the of which do not the of underlying reflexes or motor mechanisms to an when the mechanisms of paracrine of intrinsic neuronal reflexes are are no of to was for tension pellet propulsion, analysis and and to tension and pellet propulsion and of the the analysis and of and the TPH1KO mice, and important the of the and and the and the This was by from the of and was in a by a of
Heredia et al. (Tue,) studied this question.