Spontaneous Colonic Migrating Motor Complex Research Articles

Use of Genetically Encoded Calcium Indicators (GECIs) Combined with Advanced Motion Tracking Techniques to Examine the Behavior of Neurons and Glia in the Enteric Nervous System of the Intact Murine Colon.

Genetically encoded Ca2+ indicators (GECIs) have been used extensively in many body systems to detect Ca2+ transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca2+ indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We bred several GCaMP3 mice: (1) Wnt1-GCaMP3, all enteric neurons and glia; (2) GFAP-GCaMP3, enteric glia; (3) nNOS-GaMP3, enteric nitrergic neurons; and (4) ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC), using low power Ca2+ imaging. In this preliminary study, we observed neuronal and glial cell Ca2+ transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100–1000 active cells) through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca2+ transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca2+ transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca2+ transients in some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural activity but continued long after neural activity had waned. With these new tools an unprecedented level of detail can be recorded from the enteric nervous system (ENS) with minimal manipulation of tissue. These techniques can be extended in order to better understand the roles of particular enteric neurons and glia during normal and disordered motility.

Rebuttal from Terence K. Smith and Michael D. Gershon.

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 (Bulbring et al. 1958) and verified (Frigo & Lecchini, 1970). Bulbring 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.

The Nav1.9 channel regulates colonic motility in mice

The colonic migrating motor complex (CMMC) is a major pattern of motility that is entirely generated and organized by the enteric nervous system. We have previously demonstrated that the Nav1.9 channel underlies a tetrodotoxin-resistant sodium current which modulates the excitability of enteric neurons. The aim of this study was to observe the effect of loss of the Nav1.9 channel in enteric neurons on mouse colonic motility in vitro. The mechanical activity of the circular muscle was simultaneously recorded from three sites, namely, proximal, mid- and distal, along the whole colon of male, age-matched wild-type and Nav1.9 null mice. Spontaneous CMMCs were observed in all preparations. The mean frequency of CMMCs was significantly higher in the Nav1.9 null mice (one every 2.87 ± 0.1 min compared to one every 3.96 ± 0.23 min in the wild type). The mean duration of CMMCs was shorter and the mean area-under-contraction was larger in the Nav1.9 null mice compared to the wild type. In addition, CMMCs propagated preferentially in an aboral direction in the Nav1.9 null mice. Our study demonstrates that CMMCs do occur in mice lacking the Nav1.9 channel, but their characteristics are significantly different from controls. Up to now, the Nav1.9 channel was mainly associated with nociceptive neurons and involved in their hyperexcitability after inflammation. Our result shows for the first time a role for the Nav1.9 channel in a complex colonic motor pattern.

Firing patterns and functional roles of different classes of spinal afferents in rectal nerves during colonic migrating motor complexes in mouse colon

The functional role of the different classes of visceral afferents that innervate the large intestine is poorly understood. Recent evidence suggests that low-threshold, wide-dynamic-range rectal afferents play an important role in the detection and transmission of visceral pain induced by noxious colorectal distension in mice. However, it is not clear which classes of spinal afferents are activated during naturally occurring colonic motor patterns or during intense contractions of the gut smooth muscle. We developed an in vitro colorectum preparation to test how the major classes of rectal afferents are activated during spontaneous colonic migrating motor complex (CMMC) or pharmacologically induced contraction. During CMMCs, circular muscle contractions increased firing in low-threshold, wide-dynamic-range muscular afferents and muscular-mucosal afferents, which generated a mean firing rate of 1.53 ± 0.23 Hz (n = 8) under isotonic conditions and 2.52 ± 0.36 Hz (n = 17) under isometric conditions. These low-threshold rectal afferents were reliably activated by low levels of circumferential stretch induced by increases in length (1-2 mm) or load (1-3 g). In a small proportion of cases (5 of 34 units), some low-threshold muscular and muscular-mucosal afferents decreased their firing rate during the peak of the CMMC contractions. High-threshold afferents were never activated during spontaneous CMMC contractions or tonic contractions induced by bethanechol (100 μM). High-threshold rectal afferents were only activated by intense levels of circumferential stretch (10-20 g). These results show that, in the rectal nerves of mice, low-threshold, wide-dynamic-range muscular and muscular-mucosal afferents are excited during contraction of the circular muscle that occurs during spontaneous CMMCs. No activation of high-threshold rectal afferents was detected during CMMCs or intense contractile activity in naïve mouse colorectum.

Localization of the Sensory Neurons and Mechanoreceptors Required for Stretch-Evoked Colonic Migrating Motor Complexes in Mouse Colon

The pacemaker and pattern generator that underlies the cyclical generation of spontaneous colonic migrating motor complexes (CMMCs) has recently been identified to lie within the myenteric plexus and/or muscularis externa. Neither the mucosa, nor the release of substances from the mucosa were found to be required for the spontaneous generation of CMMCs. However, it is known that stretch applied to the colonic wall can also evoke CMMCs and since stretch of the gut wall is known to stimulate the mucosa, it is not clear whether release of substances from the mucosa and/or submucosal plexus are required for stretch-evoked CMMCs. Therefore, the aim of this study was to determine whether circumferential stretch-evoked CMMCs require the presence of the mucosa and/or submucosal plexus in isolated mouse colon. Spontaneous CMMCs were recorded from full length sheet preparations of colon in vitro. Graded circumferential stretch (at a rate of 100 μm/s) applied to a 15-mm segment of mid–distal colon reliably evoked a CMMC, which propagated to the oral recording site. Sharp dissection to remove the mucosa and submucosal plexus from the entire colon did not prevent spontaneous CMMCs and circumferential stretch-evoked CMMCs were still reliably evoked by circumferential stretch, even at significantly lower thresholds. In contrast, in intact preparations, direct stimulation of the mucosa (without accompanying stretch) proved highly inconsistent and rarely evoked a CMMC. These observations lead to the inescapable conclusion that the sensory neurons activated by colonic stretch to initiate CMMCs lie in the myenteric plexus, while the mechanoreceptors activated by stretch, lie in the myenteric ganglia and/or muscularis externa. Stretch activation of these mechanoreceptors does not require release of any substance(s) from the mucosa, or neural inputs arising from submucosal ganglia.

Calcium activity in different classes of myenteric neurons underlying the migrating motor complex in the murine colon

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.

Potentiation of a functional autoantibody in narcolepsy by a cholinesterase inhibitor

We have recently reported the presence of an immunoglobulin G (IgG) autoantibody (Ab) in patients with narcolepsy with cataplexy that abolishes spontaneous colonic migrating motor complexes (CMMCs) and increases smooth muscle tension and atropine-sensitive phasic contractions in a physiological assay of an isolated colon. In this study, we used the cholinesterase inhibitor, neostigmine, to explore the mechanism of the narcoleptic IgG-mediated disruption of enteric motor function in four patients with narcolepsy with cataplexy and to identify a pharmacological mimic of the Ab. Neostigmine potentiated the narcoleptic IgG-mediated increase in smooth muscle resting tension and phasic smooth muscle contractions by an atropine-sensitive mechanism but exerted no effect on resting tension in the presence of control IgG. Decreased frequency of CMMCs mediated by IgG with anti-M3R activity was reversed by neostigmine. Therefore, a challenge with a cholinesterase inhibitor improves the specificity of the CMMC assay for narcoleptic IgG. Tetrodotoxin (TTX), a neuronal sodium channel blocker, also abolished CMMCs and increased resting tone, and a similar potentiation was observed with neostigmine; thus, TTX is a mimic of the functional effects of the narcoleptic IgG in this bioassay. These findings provide a link to pharmacological studies of canine narcolepsy and are consistent with a functional blockade of both excitatory and inhibitory motor neurons by the narcoleptic Ab, similar to the TTX mimic, presumably by binding to an autoantigenic target expressed in both populations of neurons.

Release of 5-Hydroxytryptamine From the Mucosa Is Not Required for the Generation or Propagation of Colonic Migrating Motor Complexes

The pacemaker mechanism that underlies the cyclic generation of colonic migrating motor complexes (CMMCs) is unknown, although studies have suggested that release of 5-hydroxytryptamine (5-HT) from enterochromaffin cells in the mucosa is essential. However, no recordings of 5-HT release from the colon have been made to support these suggestions. We used real-time amperometry to record 5-HT release directly from the mucosa in mouse isolated colon to determine whether 5-HT release from enterochromaffin cells was required for CMMC generation. We found that 5-HT was released from mucosal enterochromaffin cells during many, but not all, CMMC contractions. However, spontaneous CMMCs still were recorded even after removal of the mucosa, and submucosa and submucosal plexus when all release of 5-HT had been abolished. CMMC pacemaker frequency was slower in the absence of the mucosa, an effect reversed by focal application of exogenous 5-HT onto the myenteric plexus. Despite the absence of the mucosa and all detectable release of 5-HT, ondansetron significantly reduced CMMC frequency, suggesting that 5-HT(3) receptor blockade slows the CMMC pacemaker via a mechanism independent of 5-HT release from enterochromaffin cells. Our results show that 5-HT can be released dynamically during CMMCs. However, the intrinsic pacemaker and pattern generator underlying CMMC generation lies within the myenteric plexus and/or muscularis externa and does not require any release of 5-HT from enterochromaffin cells. Endogenous release of 5-HT from enterochromaffin cells plays a modulatory role, not an essential role, in CMMC generation.

An Autoantibody in Narcolepsy Disrupts Colonic Migrating Motor Complexes

Despite strong circumstantial evidence for the autoimmune hypothesis of narcolepsy, conventional immunological methods have failed to detect an autoantibody. This study investigated the real-time effects of narcoleptic immunoglobulins on a spontaneous colonic migrating motor complex (CMMC) preparation. IgG from patients with narcolepsy with cataplexy or healthy controls was added directly to isolated mouse colons undergoing CMMC activity to test for autoantibodies that disrupt colonic motility. The effect of immunoglobulins prepared for clinical intravenous treatment (IVIg) on autoantibody-mediated colonic disruption was also assessed. Narcoleptic IgGs markedly reduced the frequency of CMMCs or irreversibly abolished them. Abrogation of CMMCs was followed by an increase in the resting tension of the colon preparation and appearance of atropine-sensitive phasic smooth muscle contractions. IVIg partially neutralized the inhibitory effect of narcoleptic IgG on the CMMCs. The dramatic effect of narcoleptic IgG on CMMC generation is consistent with an autoantibody-mediated disruption of enteric neural pathways. The ex vivo whole-organ approach allows real-time examination of the physiological effects of the narcoleptic autoantibody and offers a new avenue for exploring the autoimmune basis of narcolepsy. The neutralizing effect of IVIg on the autoantibody provides a rationale for the reported clinical improvement in cataplexy when IVIg are given at disease onset.

Motility in the isolated mouse colon: migrating motor complexes, myoelectric complexes and pressure waves.

This study has used mechanical, together with pressure/volume recordings or electrophysiological recordings, to investigate the spontaneous activity in isolated preparations of mouse colon. In the former preparations, when not distended with fluid, spontaneous colonic migrating motor complexes (CMMCs) were observed using isotonic transducers. When the colons were distended with fluid, CMMCs continued at an increased frequency and in addition were associated temporally, with rises in intraluminal pressure and pulses of distally ejected fluid. 5-Hydroxytryptamine (1 micro mol L-1) or NG-nitro-l-arginine (100 micro mol L-1) increased the frequency of propulsive activity and this activity was abolished by hexamethonium (500 micro mol L-1). In a second preparation, myoelectric complexes recorded from circular muscle cells in colons using intracellular microelectrodes, were found to correlate in frequency and phase with CMMCs. The experiments indicate that CMMCs are intimately related to pressure waves in the fluid-filled viscus and the muscle membrane potential changes that have been recorded during myoelectric complexes are likely to be analogous to those occurring during fluid-filled propulsive activity.

Neural integrity is essential for the propagation of colonic migrating motor complexes in the mouse.

The mechanisms that underlie the propagation of contractions along the colon are uncertain. We have examined whether spontaneous colonic migrating motor complexes (CMMCs) migrate through a region of muscle paralysis, or through a region where neural transmission was disrupted in the isolated mouse colon. Mouse colon was mounted in a separately perfused three-compartment organ bath and recordings of circular muscle tension were made. Drug application was restricted to the middle compartment. Application of nifedipine (1 micromol L(-1)), an l-type calcium channel antagonist, reduced the contraction amplitude by approximately 94%, without affecting the form of contractions in the proximal and distal compartments. Moreover, CMMCs appeared to remain temporally related in all compartments. In contrast, interruption of neural transmission in the middle compartment by either tetrodotoxin (1.6 micromol L(-1)), hexamethonium (500 micromol L(-1)) or a low-calcium, high-magnesium solution abolished CMMCs in this compartment; contractions recorded in the proximal and distal compartments became slower in frequency and were no longer synchronized. The experiments suggest that there may be more than one 'pacemaker' generating spontaneous CMMCs and that CMMCs can migrate through a region where there is minimal tension generation, but not through a region where neural integrity has been compromised.

Endogenous nitric oxide release modulates the direction and frequency of colonic migrating motor complexes in the isolated mouse colon

Spontaneous colonic migrating motor complexes (CMMCs) were recorded from circular muscle at three sites along the isolated mouse colon. The interval between CMMCs was decreased from approximately 3 min in control solution, by approximately 55% in a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (L-NNA; 100 micromol L-1). This was associated with a shift in migration direction of CMMCs, such that CMMCs migrated in an oral direction. Application of the endogenous substrate for NOS, L-arginine, at a low concentration used to mimic plasma concentration (134 micromol L-1), or a high concentration (5 mmol L-1) suppressed CMMCs (for at least 15 min) which were replaced by high frequency (10-15 min-1), short duration (half width approximately 1.5 s) contractions of variable amplitudes (largest in the proximal region) until CMMCs resumed. CMMCs remained in the presence of D-arginine (134 micromol L-1 and 5 mmol L-1). Apamin (250 nmol L-1) did not alter the interval between CMMCs, however, additional nonmigrating contractions were observed between the CMMCs in the distal region. In addition to its effects on smooth muscle tone, NO, but not apamin-sensitive channels, plays an important role in suppressing the frequency of migrating contractions in the isolated mouse colon. Consideration should be given to the inclusion of L-arginine, in in vitro experiments where there may be spontaneous activity in NOS containing neurones.