Background & Aims-The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter, through a rhythmic sequence of electrical activity and/or contractions, along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity.

The spontaneous colonic migrating motor complex (CMMC) is a cyclical contractile and electrical event that is the primary motor pattern underlying fecal pellet propulsion along the murine colon. We have combined Ca 2+ imaging with immunohistochemistry to determine the role of different classes of myenteric neurons during the CMMC. Between CMMCs, myenteric neurons usually displayed ongoing but uncoordinated activity. Stroking the mucosa at the oral or anal end of the colon resulted in a CMMC (latency: ∼6 to 10 s; duration: ∼28 s) that consisted of prolonged increases in activity in many myenteric neurons that was correlated to Ca 2+ transients in and displacement of the muscle. These neurons were likely excitatory motor neurons. Activity in individual neurons during the CMMC was similar regardless of whether the CMMC occurred spontaneously or was evoked by anal or oral mucosal stimulation. This suggests that convergent interneuronal pathways exist which generate CMMCs. Interestingly, Ca 2+ transients in a subset of NOS +ve neurons were substantially reduced during the CMMC. These neurons are likely to be inhibitory motor neurons that reduce their activity during a complex (disinhibition) to allow full excitation of the muscle. Local stimulation of the mucosa evoked synchronized Ca 2+ transients in Dogiel Type II (mitotracker/calbindin-positive) neurons after a short delay (∼1-2 s), indicating they were the sensory neurons underlying the CMMC. These local responses were observed in hexamethonium, but were blocked by ondansetron (5-HT 3 antagonist), suggesting Dogiel Type II neurons were activated by 5-HT release from enterochromaffin cells in the mucosa. In fact, removal of the mucosa yielded no spontaneous CMMCs, although many neurons (NOS +ve and NOS −ve) exhibited ongoing activity, including Dogiel Type II neurons. These results suggest that spontaneous or evoked 5-HT release from the mucosa is necessary for the activation of Dogiel Type II neurons that generate CMMCs.

Simultaneous intracellular recordings were made from myenteric neurons and circular muscle (CM) cells in isolated, stretched segments of guinea-pig distal colon. We have shown previously that maintained stretch generates a repetitive and coordinated discharge of ascending excitatory and descending inhibitory neuronal reflex pathways in the distal colon. In the presence of nifedipine (1-2 µM) to paralyse the muscle, simultaneous recordings were made from 25 pairs of AH (after-hyperpolarization)-neurons and CM cells separated by 100-500 µm. In all 25 AH-neurons, proximal process potentials (PPPs) were never recorded, even though at the same time, all recordings from neighbouring CM cells showed an ongoing discharge of inhibitory junction potentials (IJPs) anally, or excitatory junction potentials (EJPs) orally. In fact, 24 of 25 AH-neurons were totally silent, while in one AH-cell, some spontaneous fast excitatory postsynaptic potentials (FEPSPs) were recorded. All 10 electrically silent AH-cells that were injected with neurobiotin were found to be multipolar Dogiel type II neurons. In contrast, when recordings were made from myenteric S-neurons, two distinct electrical patterns of electrical activity were recorded. Recordings from 25 of 48 S-neurons showed spontaneous FEPSPs, the majority of which (22 of 25) showed periodswhendiscreteclustersof FEPSPs(meanduration88 ms)couldbetemporallycorrelated with the onset of EJPs or anal IJPs in the CM. Nine S-neurons were electrically quiescent. The second distinct electrical pattern in 14 S-neurons consisted of bursts, or prolonged trains of action potentials, which could be reduced to proximal process potentials (PPPs) in six of these 14 neurons during membrane hyperpolarization. Unlike FEPSPs, PPPs were resistant to a low Ca 2+ -high Mg 2+ solution and did not change in amplitude during hyperpolarizing pulses. Mechanosensory S-neurons were found to be uniaxonal or pseudounipolar filamentous neurons, with morphologies consistent with interneurons. No slow EPSPs were ever recorded from AH-or S-type neurons when IJPs or EJPs occurred in the CM. In summary, we have identified a population of mechanosensory S-neurons in the myenteric plexus of the distal colon which appear to be largely stretch sensitive, rather than muscle-tension sensitive, since they generate ongoing trains of action potentials in the presence of nifedipine. No evidence was found to suggest that in paralysed preparations, the repetitive firing in ascending excitatory or descending inhibitory nerve pathways was initiated by myenteric AH-neurons, or slow synaptic transmission.

Key points• 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-HT 3 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) 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-HT 3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1 +/+ mice, failed to alter CMMCs in Tph1 −/− animals. Our observations suggest ...

Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase (nNOS)(-/-) mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and nNOS(-/-) mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 +/- 0.6 mV; nNOS(-/-): 11.2 +/- 1.5 mV). Both atropine (1 microM) and MEN 10,376 [neurokinin 2 (NK2) antagonist; 0.5 microM] added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 microM) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted nNOS(-/-) mouse colon (wild-type: 47.9 +/- 2.4 mm; nNOS(-/-): 57.8 +/- 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks.

Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca2+ imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca2+ transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca2+ transient wave fronts propagated through the ICC-MY networks at ∼2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca2+ transients in LM but had no visible effect on the transients in ICC-MY. β-Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca2+ entry (Ni2+ and mibefradil) and elevated external K+ reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca2+ transients in ICC-MY. These data show that global Ca2+ transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca2+ handling mechanisms and entrained by voltage-dependent Ca2+ entry and coupling of cells via gap junctions.

Dickson EJ, Heredia DJ, Smith TK. Critical role of 5-HT1A, 5-HT 3, and 5-HT7 receptor subtypes in the initiation, generation, and propagation of the murine colonic migrating motor complex. Am J Physiol Gastrointest Liver Physiol 299: G144 -G157, 2010. First published April 22, 2010; doi:10.1152/ajpgi.00496.2009.-The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT 3 receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT 1A, 5-HT3, and 5-HT 7 receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 M; 5-HT 3 antagonist) plus hexamethonium (100 M) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 M) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 M; 5-HT 7 antagonists). Both and (S)-WAY100135 (1-5 M; 5-HT 1A antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 M), and 5-carboxamidotryptamine (5-CT) (5 M; 5-HT 1/5/7 agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT1A, 5-HT3, and 5-HT7 receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT7 receptors on AH neurons these interneurons also contribute to the generation of the CMMC.5-hydroxytryptamine; AH neurons; cholinergic transmission; circular muscle; enterochromaffin cells; mucosa; serotonergic transmission; smooth muscle OVER 95% OF THE SEROTONIN IN THE BODY, also called 5-hydroxytryptamine (5-HT), resides in the gastrointestinal tract, where it plays an important role in regulating gut motility, secretion, sensation, and bone metabolism (6,15,16,45,46). Most of this 5-HT is in enterochromaffin (EC) cells in the mucosa although it is also present in a small population (ϳ1%) of descending myenteric interneurons that project to the submucus plexus, suggesting they may modulate secretomotor reflexes. Secretomotor reflexes and motility reflexes can be activated by 5-HT release from EC cells (5,6...

The distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500 +/- 100 reactive nerve cells per cm2 of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiel's type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.