Gastroparesis is a motility disorder of the upper gastrointestinal tract in the setting of delayed stomach emptying. Gastroparesis presents with refractory nausea and vomiting, early satiety, bloating as well as abdominal pain. The pathophysiology of gastroparesis is not clear. Recent studies have investigated the histological changes of the stomach which may contribute to the development of gastroparesis. This chapter highlights the pathological findings of gastroparesis with a major focus on interstitial cells of Cajal (ICC) loss, fibrosis and enteric neuronal defects.

The enteric nervous system in the large intestine generates two important patterns relating to motility: 1) propagating rhythmic peristaltic smooth muscle contractions referred to as colonic migrating motor complexes (CMMCs) and 2) tonic inhibition, during which colonic smooth muscle contractions are suppressed. The precise neurobiological substrates underlying each of these patterns are unclear. Using transgenic animals expressing the genetically encoded calcium indicator GCaMP3 to monitor activity or the optogenetic actuator channelrhodopsin (ChR2) to drive activity in defined enteric neuronal subpopulations, we provide evidence that cholinergic and nitrergic neurons play significant roles in mediating CMMCs and tonic inhibition, respectively. Nitrergic neurons [neuronal nitric oxide synthase (nNOS)-positive neurons] expressing GCaMP3 exhibited higher levels of activity during periods of tonic inhibition than during CMMCs. Consistent with these findings, optogenetic activation of ChR2 in nitrergic neurons depressed ongoing CMMCs. Conversely, cholinergic neurons [choline acetyltransferase (ChAT)-positive neurons] expressing GCaMP3 markedly increased their activity during the CMMC. Treatment with the NO synthesis inhibitor Nω-nitro-l-arginine also augmented the activity of ChAT-GCaMP3 neurons, suggesting that the reciprocal patterns of activity exhibited by nitrergic and cholinergic enteric neurons during distinct phases of colonic motility may be related.NEW & NOTEWORTHY Correlating the activity of neuronal populations in the myenteric plexus to distinct periods of gastrointestinal motility is complicated by the difficulty of measuring the activity of specific neuronal subtypes. Here, using mice expressing genetically encoded calcium indicators or the optical actuator channelrhodopsin-2, we provide compelling evidence that cholinergic and nitrergic neurons play important roles in mediating coordinated propagating peristaltic contractions or tonic inhibition, respectively, in the murine colon.

Protocol for harvest of colonic intestinal tissue, with the intent of imaging the myenteric plexus.

We discuss the role of multiple cell types involved in rhythmic motor patterns in the large intestine that include tonic inhibition of the muscle layers interrupted by rhythmic colonic migrating motor complexes (CMMCs) and secretomotor activity. We propose a model that assumes these motor patterns are dependent on myenteric descending 5-hydroxytryptamine (5-HT, serotonin) interneurons. Asynchronous firing in 5-HT neurons excite inhibitory motor neurons (IMNs) to generate tonic inhibition occurring between CMMCs. IMNs release mainly nitric oxide (NO) to inhibit the muscle, intrinsic primary afferent neurons (IPANs), glial cells, and pacemaker myenteric pacemaker interstitial cells of Cajal (ICC-MY). Mucosal release of 5-HT from enterochromaffin (EC) cells excites the mucosal endings of IPANs that synapse with 5-HT descending interneurons and perhaps ascending interneurons, thereby coupling EC cell 5-HT to myenteric 5-HT neurons, synchronizing their activity. Synchronized 5-HT neurons generate a slow excitatory postsynaptic potential in IPANs via 5-HT 7 receptors and excite glial cells and ascending excitatory nerve pathways that are normally inhibited by NO. Excited glial cells release prostaglandins to inhibit IMNs (disinhibition) to allow full excitation of ICC-MY and muscle by excitatory motor neurons (EMNs). EMNs release ACh and tachykinins to excite pacemaker ICC-MY and muscle, leading to the simultaneous contraction of both the longitudinal and circular muscle layers. Myenteric 5-HT neurons also project to the submucous plexus to couple motility with secretion, especially during a CMMC. Glial cells are necessary for switching between different colonic motor behaviors. This model emphasizes the importance of myenteric 5-HT neurons and the likely consequence of their coupling and uncoupling to mucosal 5-HT by IPANs during colonic motor behaviors.

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

The question at hand is whether 5-HT is necessary for peristalsis. Smith & Gershon (2015) have marshalled experimental evidence that makes a compelling case for the necessity of 5-HT. Spencer et al. (2015) rebut none of that evidence but advance an argument that refutes a straw man of assertions that no one has made. 5-HT receptors are arbitrarily assigned constitutive activity so that antagonists can be called inverse agonists with no supporting data. Spencer el al. also use an ad hominem attack to denigrate contrary observations which are dismissed as the result of an inadequate number of experiments that have been performed incorrectly. In fact, the mucosa was removed from over 80 preparations without ever observing a spontaneous colonic migrating motor complex (CMMC), yet ongoing neural activity of the myenteric plexus was preserved (Heredia et al. 2009; Bayguinov et al. 2010; Dickson et al. 2010). Moreover, it is not new that mucosal removal, or mucosal asphyxiation or anaesthetization, abolishes peristaltic reflexes in guinea pigs, rabbits and cats (Bülbring et al. 1958) and verified (Frigo & Lecchini, 1970). Bülbring proposed that enterochromaffin (EC) cells secrete 5-HT, which stimulates sensory (primary afferent) neurons to initiate peristaltic reflexes, and predicted that prevention of mucosal 5-HT biosynthesis would impair peristaltic reflexes. In fact, despite the incorrect contrary assertion of Spencer et al. (2015), it does (Heredia et al. 2013). Brushing of the mucosal surface elicits CMMCs in isolated wild-type colon but not in that of mice lacking tryptophan hydroxylase 1 (TPH1KO mice) and thus mucosal 5-HT. To evoke CMMCs in the absence of mucosal 5-HT, it is necessary to dilate/stretch the gut, which short circuits the mucosa and activates mechanosensitive neurons (Smith et al. 2010). In the TPH1KO colon small pellets, which fail to dilate the gut, are not propelled. Although fecal pellets form in situ in the TPH1KO colon they are oversized and the colon is elongated and dilated, illustrating the pathophysiological toll exacted by the absence of mucosal 5-HT. Equally important, motility is a direct function of luminal bacteria that regulate 5-HT biosynthesis in EC cells (Yano et al. 2015). Altering mucosal 5-HT biosynthesis (Brown et al. 2011) or actions (Chey et al. 2015) has, furthermore, proven to be effective therapeutically in treating intestinal motility disorders. Mucosal 5-HT might not be the only driver of peristalsis since 5-HT neurons are still present, but it is a necessary one that provides insights into GI physiology, pathophysiology and therapy. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘Last Word’. Please email your comment, including a title and a declaration of interest to jphysiol@physoc.org. Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None declared.

The overwhelming preponderance of the 5-HT of every known mammal is in the gut (Erspamer, 1966; Gershon & Tack, 2007; Gershon, 2013). Enteric 5-HT must be an important signalling molecule to be so conserved. Most enteric 5-HT is in enterochromaffin (EC) cells, but smaller amounts are present in myenteric neurons. Despite their small numbers, serotonergic neurons project widely throughout the enteric nervous system (ENS) and also innervate interstitial cells of Cajal (ICC) (Okamoto et al. 2014). The abundance and variety of enteric 5-HT receptors (5-HT1-7 and subtypes) also suggest that 5-HT plays a significant role in GI physiology (Smith et al. 2014). The efficacy of therapies targeting enteric 5-HT or its receptors against GI motility disorders supports this idea (Gershon & Tack, 2007; Gershon, 2013). Beyond motility and secretion, putative roles that 5-HT plays include metabolism, osteogenesis, immunity, neurogenesis and neuroprotection (Gershon, 2013). The multiplicity of enteric 5-HT targets and receptors complicates ascertaining the physiological roles of 5-HT. Controversy is thus to be expected and has appeared in recent papers, which question whether EC or neuronal 5-HT has anything to do with peristalsis (Keating & Spencer, 2010; Spencer et al. 2011; Sia et al. 2013; Spencer et al. 2013). These papers are important not because enteric 5-HT is vestigial (it is not) or that its roles in normal and abnormal GI motility can be ignored (they cannot). Instead, they highlight common misunderstandings about peristaltic reflexes and 5-HT cellular biology. The papers focus on colonic migrating motor complexes (CMMCs), which are aborally propagating propulsive contractile complexes, essentially peristaltic reflexes. The authors assert that they can evoke CMMCs after mucosal removal or depleting 5-HT with reserpine; therefore, they conclude that neither EC cells, nor neuronal 5-HT is necessary for CMMCs. To comprehend what is misunderstood, it is necessary to discuss basic information about 5-HT and GI motility. Two tryptophan hydroxylase isoforms, TPH1 and TPH2, are rate limiting in 5-HT biosynthesis, TPH1 in EC cells, and TPH2 in serotonergic neurons (Gershon, 2013). 5-HT is synthesized in the cytosol but stored in vesicles. Reuptake terminates actions of 5-HT. Because 5-HT is charged, two transporters are required for transmembrane transport, a vesicular monoamine transporter (VMAT1 in EC cells and VMAT2 in neurons) (Henry et al. 1998) and a plasmalemmal serotonin reuptake transporter (SERT) (Blakely, 2001). Reserpine inhibits VMAT (Henry et al. 1998). Intracellular 5-HT is thus reduced due to enhanced catabolism; but reserpine does not prevent 5-HT biosynthesis or constitutive release. That requires deletion or inhibition of TPH, which when isoform-selective, distinguishes mucosal from neuronal 5-HT (Li et al. 2011; Gershon, 2013). SERT deletion amplifies 5-HT effects. Because GI motility is abnormal after deletion of SERT (Chen et al. 2001), either isoform of TPH (Li et al. 2011; Gershon, 2013), or exposure to 5-HT antagonists/agonists (Monro et al. 2002; Smith et al. 2014), 5-HT clearly influences GI motility. Peristalsis is a general term applied to enteric motile behaviour that should not be conflated with the ENS-mediated peristaltic reflex that can drive propulsion (Gershon & Tack, 2007; Gershon, 2013; Furness et al. 2014). That reflex, first called the 'law of the intestine' (Bayliss & Starling, 1899), is an oral contraction and anal relaxation; it is evoked by increased intraluminal pressure and involves polarized neural pathways within the ENS (Furness et al. 2014; Smith et al. 2014). Many enteric cells, not just neurons and muscle, participate in peristaltic reflexes (Smith et al. 2014). The peristaltic reflex is only one of many activity patterns encoded within the ENS (Furness et al. 2014). Mucosal pressure/distortion or chemical stimuli release 5-HT from EC cells and evoke peristaltic reflexes (Bülbring & Lin, 1958; Bertrand et al. 2008). Luminally applied 5-HT mimics pressure (Bülbring & Crema, 1958; Bülbring & Lin, 1958). Fecal pellets apply pressure to the mucosa and thereby release 5-HT, which entrains CMMCs (Heredia et al. 2009). Mucosal removal, anaesthesia, or asphyxiation all abolish mucosally evoked peristaltic reflexes (Bülbring & Crema, 1958; Bayguinov et al. 2010; Dickson et al. 2010). Mucosally released 5-HT acts on 5-HT3 and/or 5-HT1P (or 5-HT7) receptors to stimulate intrinsic primary afferent neurons (IPANs) (Pan & Gershon, 2000; Bertrand et al. 2008; Dickson et al. 2010), which engage the ENS (Kirchgessner et al. 1992; Bayguinov et al. 2010; Okamoto et al. 2014). IPANs are found in both plexuses (Kirchgessner et al. 1992; Bayguinov et al. 2010; Okamoto et al. 2014), and appear to link mucosal and neuronal 5-HT pools together (Okamoto et al. 2014; Smith et al. 2014). 5-HT3 antagonists can block CMMCs when applied around fecal pellets, as do intraluminal 5-HT3 and 5-HT7 antagonists, suggesting that these antagonists act locally on the EC cell-to-IPAN junction (Heredia et al. 2009; Smith et al. 2014). Evidence suggests that neuronal, as well as mucosal, 5-HT is critical for peristaltic reflexes. Serosally applied 5-HT desensitizes ENS receptors, thereby inhibiting peristaltic reflexes (Bülbring & Crema, 1958; Smith et al. 2014). 5-HT antagonism interferes with transmission in ENS pathways, CMMCs, and tonic inhibition in the colon (Monro et al. 2002; Dickson et al. 2010). Neuronal 5-HT can mediate intestinal slow excitatory postsynaptic potentials (sEPSPs): 5-HT7 antagonists inhibit sEPSPs in IPANs (Monro et al. 2005), as well as CMMCs (Dickson et al. 2010). Tryptamine, which first releases and then depletes endogenous 5-HT, initially induces but then abolishes sEPSPs without affecting similar responses to exogenous 5-HT (Takaki et al. 1985). Anti-idiotypic antibodies, which bind selectively to all 5-HT receptors, also mimic sEPSPs before blocking them irreversibly (Wade et al. 1994). Radial stretch of the bowel wall activates high threshold mechanosensitive interneurons that activate CMMCs (Heredia et al. 2009). Mucosal stimuli and radial stretch evoke similar reflex responses because nerve pathways from each converge on final common neurons (Smith et al. 1992, 2007). Mucosal reflexes alone propel small fecal pellets that do not produce radial stretch down the colon (Heredia et al. 2013); fluid or larger pellets that stretch the gut can be propelled in the absence of the mucosa or mucosal 5-HT (Spencer et al. 2011; Heredia et al. 2012, 2013). Because stimuli that short circuit mucosal activation evoke CMMC-like responses does not mean the mucosa and its 5-HT are not physiologically critical. Stimuli restricted to the mucosa cannot evoke CMMCs in the TPH1KO colon and thus are 5-HT-dependent (Heredia et al. 2013). If the TPH1KO bowel is stretched, CMMC-like responses are evoked; however, they do not propagate and thus are not CMMCs. In an analogy, the lower leg can be made to move involuntarily through the patellar reflex or voluntarily. Voluntary leg movement does not obviate the need for quadriceps muscle spindles to evoke patellar reflexes. Circuits in the myenteric plexus can be engaged in the absence of mucosal 5-HT to give rise to contractile activity; however, under physiological circumstances the mucosa is present and, when pressed, secretes 5-HT. When the gut is intact, therefore, 5-HT will do what it does when the mucosa releases it, initiate peristaltic reflexes. The argument (Spencer et al. 2013) that because reserpine-induced 5-HT depletion fails to prevent CMMCs, 5-HT is not needed is invalid. Because reserpine only inhibits VMAT, it cannot drive tissue 5-HT to zero. Reserpine lowers intracellular 5-HT to levels that may be difficult to detect (Bülbring & Crema, 1959; Spencer et al. 2013); however, the 5-HT that remains activates receptors. In fact, constitutive 5-HT release in reserpine-treated animals enhances intestinal motility (Bülbring & Crema, 1959). The continued secretion of 5-HT thus explains the ability of 5-HT3 and 5-HT4 antagonists to block responses in reserpinized preparations (Sia et al. 2013; Spencer et al. 2013). Importantly, 5-HT3 antagonists do not affect the CMMC-like activity in TPH1KO mice (Heredia et al. 2013). The abnormality of CMMCs in TPH1KO mice establishes that physiologically meaningful peristaltic reflexes are 5-HT dependent (Heredia et al. 2013). Total GI transit and colonic motility are slowed in TPH2KO mice but gastric emptying is accelerated (Li et al. 2011; Gershon, 2013). Normal GI motility thus requires neuronal 5-HT; however, because neuronal 5-HT is a growth factor, the TPH2KO ENS is severely hypoplastic (Li et al. 2011), which could be responsible for defective GI motility. Accelerated gastric emptying probably occurs because serotonergic activation of gastric inhibitory motor neurons is impaired in mice lacking neuronal 5-HT (Li et al. 2011). Patterns of GI motility other than peristaltic reflexes evidently compensate for the defective peristaltic reflex/CMMC of TPH1KO mice (Li et al. 2011; Gershon, 2013). Compensatory mechanisms, such as enhanced prostaglandin synthesis may also be mobilized to generate CMMC-like activity in the absence of mucosal 5-HT (Heredia et al. 2012; Smith et al. 2014). In conclusion, 5-HT has satisfied all of the criteria needed to identify it a mediator of peristaltic reflexes. EC cells and enteric neurons synthesize 5-HT. Exogenous 5-HT and stimuli that release endogenous 5-HT evoke peristaltic reflexes, which are lost or impaired when mucosal and/or neuronal 5-HT is depleted or 5-HT receptors are antagonized. Mucosal and neuronal 5-HT are thus essential for physiological manifestation of peristaltic reflexes. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a 'Last Word'. Please email your comment, including a title and a declaration of interest to jphysiol@physoc.org. Comments will be moderated and accepted comments will be published online only as 'supporting information' to the original debate articles once discussion has closed. Terence Smith (right) is Professor in the Department of Physiology and Cell Physiology at the University of Nevada-Reno, USA, where he is the Director of the Dynamic Imaging Core. After working for several years in solid state physics, he received his PhD in neuropharmacology/electrophysiology from Monash University, Victoria, Australia under Professors Mollie Holman and David Hirst. His interests have focused on the circuitry in the enteric nervous system and how this affects gut pacemakers. Michael Gershon (left) is Professor of Pathology and Cell Biology at Columbia University, College of Physicians and Surgeons. He received his MD degree from Cornell University, did post-doctoral research with Professor Edith Bülbring in the Department of Pharmacology of Oxford University, and chaired the Department of Anatomy and Cell Biology at Columbia until 2006. His interests are in enteric neuronal development, cell biology, and function as well as the roles serotonin plays in the bowel. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None declared. This work was funded by grants from the National Institutes of Health: RO1 DK45713 (T.K.S.) and NS 12969, NS 15547 (M.D.G.).

The colonic migrating motor complex (CMMC) is a critical neurally mediated rhythmic propulsive contraction observed in the large intestine of many mammals. It seems to be equivalent to the high amplitude propagating contractions (HAPCs) in humans. This review focuses on the probable neural mechanisms involved in producing the CMMC or HAPC, their likely de-pendence on mucosal and neuronal serotonin and pacemaker insterstitial cells of Cajal networks and how intrinsic neural re-flexes affect them. Discussed is the possibility that myenteric 5-hydroxytryptamine (5-HT) neurons are not only involved in tonic inhibition of the colon, but are also involved in generating the CMMC and modulation of the entire enteric nervous system, including coupling motility to secretion and blood flow. Mucosal 5-HT appears to be important for the initiation and effective propagation of CMMCs, although this mechanism is a longstanding controversy since the 1950s, which we will address. We argue that the slow apparent propagation of the CMMC/HAPC down the colon is unlikely to result from a slowly conducting wave front of neural activity, but more likely because of an interaction between ascending excitatory and descending (serotonergic) inhibitory neural pathways interacting both within the myenteric plexus and at the level of the muscle. That is, CMMC/HAPC propagation appears to be similar to esophageal peristalsis. The suppression of inhibitory (neuronal nitric oxide synthase) motor neurons and mucosal 5-HT release by an upregulation of prostaglandins has important implications in a num-ber of gastrointestinal disorders, especially slow transit constipation.(J Neurogastroenterol Motil 2014;20:423-446).

Background5-Hydroxytryptamine (5-HT, serotonin) is an important regulator of colonic motility and secretion; yet the role of serotonergic neurons in the colon is controversial.MethodsWe used immunohistochemical techniques to examine their projections throughout the enteric nervous system and interstitial cells of Cajal (ICC) networks in the murine proximal to mid colon.Key ResultsSerotonergic neurons, which were mainly calbindin positive, occurred only in myenteric ganglia (1 per 3 ganglia). They were larger than nNOS neurons but similar in size to Dogiel Type II (AH) neurons. 5-HT neurons, appeared to make numerous varicose contacts with each other, most nNOS neurons, Dogiel Type II/AH neurons and glial cells. 5-HT, calbindin and nNOS nerve fibers also formed a thin perimuscular nerve plexus that was associated with ganglia, which contained both nNOS positive and negative neurons, which lay directly upon the submucosal pacemaker ICC network. Neurons in perimuscular ganglia were surrounded by 5-HT varicosities. Submucous ganglia contained nNOS positive and negative neurons, and calbindin positive neurons, which also appeared richly supplied by serotonergic nerve varicosities. Serotonergic nerve fibers ran along submucosal arterioles, but not veins. Varicosities of serotonergic nerve fibers were closely associated with pacemaker ICC networks and with intramuscular ICC (ICC-IM). 5-HT2B receptors were found on a subpopulation of non-5-HT containing myenteric neurons and their varicosities, pacemaker ICC-MY and ICC-IM.Conclusions & InferencesMyenteric serotonergic neurons, whose axons exhibit considerable divergence, regulate the entire enteric nervous system and are important in coordinating motility with secretion. They are not just interneurons, as regularly assumed, but possibly also motor neurons to ICC and blood vessels, and some may even be sensory neurons.

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.

The mechanisms underlying slow-transit constipation (STC) are unclear. In 50% of patients with STC, some form of outlet obstruction has been reported; also an elongated colon has been linked to patients with STC. Our aims were 1) to develop a murine model of STC induced by partial outlet obstruction and 2) to determine whether this leads to colonic elongation and, consequently, activation of the inhibitory "occult reflex," which may contribute to STC in humans. Using a purse-string suture, we physically reduced the maximal anal sphincter opening in C57BL/6 mice. After 4 days, the mice were euthanized (acutely obstructed), the suture was removed (relieved), or the suture was removed and replaced repeatedly (chronically obstructed, over 24-31 days). In partially obstructed mice, we observed increased cyclooxygenase (COX)-2 levels in muscularis and mucosa, an elongated impacted large bowel, slowed transit, nonpropagating colonic migrating motor complexes (CMMCs), a lack of mucosal reflexes, a depolarized circular muscle with slow-wave activity due to a lack of spontaneous inhibitory junction potentials, muscle hypertrophy, and CMMCs in mucosa-free preparations. Elongation of the empty obstructed colon produced a pronounced occult reflex. Removal of the obstruction or addition of a COX-2 antagonist (in vitro and in vivo) restored membrane potential, spontaneous inhibitory junction potentials, CMMC propagation, and mucosal reflexes. We conclude that partial outlet obstruction increases COX-2 leading to a hyperexcitable colon. This hyperexcitability is largely due to suppression of only descending inhibitory nerve pathways by prostaglandins. The upregulation of motility is suppressed by the occult reflex activated by colonic elongation.

Background Neuronal communication within the myenteric plexus occurs when action potentials along nerve fibers produce Ca2+ transients in varicosities leading to exocytosis of vesicles and neurotransmitters release. We used Ca2+ transients in varicosities to monitor action potential activity in myenteric nerve pathways both between and during the colonic migrating motor complex (CMMC) in the isolated murine colon. Methods Strips of longitudinal muscle were removed to reveal the myenteric ganglia, which were then loaded with Fluo-4. Key Results Many varicosities, including synaptotagmin 1 labeled varicosities, exhibited ongoing Ca2+ transients (duration of unitary Ca2+ transient 3.9 s). Between CMMCs, varicosities fired at a frequency of 0.6 Hz, which correlated with spontaneous inhibitory junction potentials in the circular muscle, suggesting they were mainly in inhibitory nerve pathways. During a CMMC other previously quiescent varicosities fired at 1.3 Hz (max. 2.0 Hz) for the duration (24 s) of the CMMC, suggesting they were on excitatory nerve pathways. Activity in varicosities was correlated with Ca2+ transient responses in a number of neurons. Some varicosities appeared to release an inhibitory neurotransmitter that reduced activity in nNOS-positive neurons. Varicosities along the same nerve fiber exhibited identical patterns of activity that allowed nerve fibers to be traced throughout the myenteric plexus and internodal strands. Activity in varicosities was reduced by hexamethonium (100 lmol L) 1), and blocked by x-conotoxin GVIA (200 nM) and tetrodotoxin (1 lmol L) 1; TTX). Conclusions & Inferences Ca2+ imaging of varicosities allows for a determination of activity in neural pathways within the enteric nervous system.