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Background: 5-HT 4 receptor (5-HT 4 R) agonists exert prokinetic actions in the GI tract, but non-selective actions and potential for stimulation of non-target 5-HT 4 Rs have limited their use. Since 5-HT 4 Rs are expressed in the colonic epithelium and their stimulation accelerates colonic propulsion in vitro, we tested whether luminally acting 5-HT 4 R agonists promote intestinal motility., Methods: Non-absorbed 5-HT 4 R agonists, based on prucalopride and naronapride, were assessed for potency at the 5-HT 4 R in vitro, and for tissue and serum distribution in vivo in mice. In vivo assessment of prokinetic potential included whole gut transit, colonic motility, fecal output, and fecal water content. Colonic motility was also studied ex vivo in mice treated in vivo. Immunofluorescence was used to evaluate receptor distribution in human intestinal mucosa., Key Results: Pharmacological screening demonstrated selectivity and potency of test agonists for 5-HT 4 R. Bioavailability studies showed negligible serum detection. Gavage of agonists caused faster whole gut transit and colonic motility, increased fecal output, and elevated fecal water content. Prokinetic actions were blocked by a 5-HT 4 R antagonist and were not detected in 5-HT 4 R knockout mice. Agonist administration promoted motility in models of constipation. Evaluation of motility patterns ex vivo revealed enhanced contractility in the middle and distal colon. Immunoreactivity for 5-HT 4 R is present in the epithelial layer of the human small and large intestines., Conclusions and Inferences: These findings demonstrated that stimulation of epithelial 5-HT 4 Rs can potentiate propulsive motility and support the concept that mucosal 5-HT 4 Rs could represent a safe and effective therapeutic target for the treatment of constipation., (© 2020 John Wiley & Sons Ltd.)

Key Points: Platelet derived growth factor receptor α (PDGFRα(+) ) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFRα(+) cells generate Ca(2+) transients in response to exogenous purines and these responses were blocked by MRS-2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca(2+) transients in PDGFRα(+) and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS-2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca(2+) transients evoked by nerve stimulation in PDGFRα(+) cells showed the same temporal characteristics as electrophysiological responses. PDGFRα(+) cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFRα(+) cells. PDGFRα(+) cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions., Abstract: Interstitial cells, known as platelet derived growth factor receptor α (PDGFRα(+) ) cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal tract (GI), but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca(2+) transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20 Hz) caused Ca(2+) transients in enteric motor nerve processes and then in PDGFRα(+) cells shortly after the onset of stimulation (latency from EFS was 280 ms at 10 Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca(2+) fluorescence just after the initiation of Ca(2+) transients in PDGFRα(+) cells. Upon cessation of EFS, several fast Ca(2+) transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca(2+) transients evoked in PDGFRα(+) cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca(2+) transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1((-/-)) mice. PDGFRα(+) cells expressed gap junction genes, and gap junction uncouplers, 18β-glycyrrhetinic acid (18β-GA) and octanol blocked Ca(2+) transients in SMCs but not in neurons or PDGFRα(+) cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFRα(+) cells by motor neurons. PDGFRα(+) cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Background: 5-Hydroxytryptamine (5-HT, serotonin) is an important regulator of colonic motility and secretion; yet the role of serotonergic neurons in the colon is controversial., Methods: We 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 Results: Serotonergic 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 & Inferences: Myenteric 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., (© 2014 John Wiley & Sons Ltd.)

An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast-like cells in the tunica muscularis many years ago, but little is known about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast-like cells express platelet-derived growth factor receptor α (PDGFRα) in mice and that antibodies for these receptors can be used to label the cells. We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast-like cells in the human colon. Fibroblast-like cells are labelled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. Platelet-derived growth factor receptor α(+) cells are distinct from c-Kit(+) interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or neuronal nitric oxide synthase (nNOS) (inhibitory motor neurons). Platelet-derived growth factor receptor α(+) cells express small conductance Ca(2+)-activated K(+) channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles. Our data suggest that PDGFRα(+) cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help develop an understanding of the role of PDGFRα(+) cells in the human GI tract in health and disease., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Colonic migrating motor complexes (CMMCs) are neurally mediated, cyclical contractile and electrical events, which typically propagate along the colon every 2-3 min in the mouse. We examined the interactions between myenteric neurons, interstitial cells of Cajal in the myenteric region (ICC-MY) and smooth muscle cells during CMMCs using Ca(2+) imaging. CMMCs occurred spontaneously or were evoked by stimulating the mucosa locally, or by brushing it at either end of the colon. Between CMMCs, most ICC-MY were often quiescent; their lack of activity was correlated with ongoing Ca(2+) transients in varicosities on the axons of presumably inhibitory motor neurons that were on or surrounded ICC-MY. Ca(2+) transients in other varicosities initiated intracellular Ca(2+) waves in adjacent ICC-MY, which were blocked by atropine, suggesting they were on the axons of excitatory motor neurons. Following TTX (1 μM), or blockade of inhibitory neurotransmission with N(ω)-nitro-L-arginine (L-NA, a NO synthesis inhibitor, 10 μM) and MRS 2500 (a P2Y(1) antagonist, 1 μM), ongoing spark/puff like activity and rhythmic intracellular Ca(2+) waves (38.1 ± 2.9 cycles min(-1)) were observed, yet this activity was uncoupled, even between ICC-MY in close apposition. During spontaneous or evoked CMMCs there was an increase in the frequency (62.9 ± 1.4 cycles min(-1)) and amplitude of Ca(2+) transients in ICC-MY and muscle, which often had synchronized activity. At the same time, activity in varicosites along excitatory and inhibitory motor nerve fibres increased and decreased respectively, leading to an overall excitation of ICC-MY. Atropine (1 μM) reduced the evoked responses in ICC-MY, and subsequent addition of an NK1 antagonist (RP 67580, 500 nM) completely blocked the responses to stimulation, as did applying these drugs in reverse order. An NKII antagonist (MEN 10,376, 500 nM) had no effect on the evoked responses in ICC-MY. Following TTX application, carbachol (1 μM), substance P (1 μM) and an NKI agonist (GR73632, 100 nM) produced the fast oscillations superimposed on a slow increase in Ca(2+) in ICC-MY, whereas SNP (an NO donor, 10 μM) abolished all activity in ICC-MY. In conclusion, ICC-MY, which are under tonic inhibition, are pacemakers whose activity can be synchronized by excitatory nerves to couple the longitudinal and circular muscles during the CMMC. ICC-MY receive excitatory input from motor neurons that release acetylcholine and tachykinins acting on muscarinic and NK1 receptors, respectively.

Background: The guinea-pig proximal colon contains semi-solid feces which are propelled by intermittent neural peristaltic waves to the distal colon, where solid pellets are formed. Between propulsive periods, complex motor patterns underlie fluid re-absorption and mixing of contents., Methods: Spatio-temporal analysis of video recordings were used to investigate neural and myogenic patterns of non-peristaltic motor activity., Key Results: At low distension (6 cmH(2)O), two major motor patterns were seen. Narrow rings of constriction (abrupt contractions) occurred at 19 cpm. These previously undescribed contractions occurred, almost simultaneously, at many points along the preparation, with a calculated propagation velocity of 110 mm s(-1). They were abolished by hexamethonium and by tetrodotoxin, indicating they were neurally mediated. Inhibition of nitric oxide synthase resulted in increased frequency of 'abrupt contractions' suggesting ongoing inhibitory modulation by endogenous nitric oxide. After tetrodotoxin, another distinct motor pattern was revealed; 'ripples'(1) consisted of shallow rings of contraction, occurring at 18 cpm and propagating at 2.7-2.9 mm s(-1) orally or aborally from multiple initiation sites. The frequency of 'ripples' increased as intraluminal pressure was raised, becoming very irregular at high distensions. L-type calcium channel blockers and openers affected the amplitude of 'ripples'. No frequency gradient of 'ripples' along the proximal colon was detected. This absence explains the multiple initiation sites which often shifted over time, and the oral and aboral propagation of 'ripples'., Conclusions & Inferences: The interaction of myogenic 'ripples' with neurogenic 'abrupt contractions' generates localized alternating rings of contractions and dilatation, well suited to effective mixing of contents.

Background: Altered calcium mobilization has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The aim of this study was to investigate the mechanisms by which disrupted intracellular Ca(2+) signalling contributes to the impaired contractility of colon circular smooth muscles., Methods: Acute colitis was induced in C57Bl/6 mice with dextran sulphate sodium (DSS) in the drinking water for 5 days., Key Results: Spontaneous and acetylcholine-evoked contractions, caffeine-evoked hyperpolarization, and SERCA2 and phospholamban expression were reduced compared with controls. Tetrodotoxin did not restore control levels of contractile activity. The amplitudes, but not the frequency, of intracellular Ca(2+) waves were increased compared with controls. Caffeine abolished intracellular Ca(2+) waves in control smooth muscle cells, but not in smooth muscle cells from DSS-treated mice. CaM kinase II activity and cytosolic levels of HDAC4 were increased, and I kappaB alpha levels were decreased in distal colon smooth muscles from DSS-treated mice., Conclusions & Inferences: These results suggest that disruptions in intracellular Ca(2+) mobilization due to down-regulation of SERCA2 and phospholamban expression lead to increased CaM kinase II activity and cytosolic HDAC4 that may contribute to the dysmotility of colonic smooth muscles in colitis by enhancing NF-kappaB 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.

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.

Background & Aims: In human and canine colon, both slow (slow waves, 2-8/min) and fast (myenteric potential oscillations [MPOs]; 16-20/min) electrical rhythms in the smooth muscle originate at the submucosal and myenteric borders, respectively. We used Ca(2+) imaging to investigate whether interstitial cells of Cajal (ICCs) at these borders generated distinct rhythms., Methods: Segments of canine colon were pinned with submucosal or myenteric surface uppermost or cut in cross section. Tissues were loaded with a Ca(2+) indicator (fluo-4), and activity was monitored at 36.5 +/- 0.5 degrees C using an electron multiplying charge coupled device (EMCCD)., Results: Rhythmic, biphasic Ca(2+) transients (5-8/min), similar in waveform to electrical slow waves, propagated without decrement as a wave front (2-5 mm/s) through the ICC-SM network lying along the submucosal surface of the circular muscle (CM). In contrast, rhythmic intracellular Ca(2+) waves (approximately 16/min) and spontaneous reductions in Ca(2+) were observed in ICCs at the myenteric border (ICC-MY). Normally, intracellular Ca(2+) waves were unsynchronized between adjacent ICC-MY, although excitatory nerve activity synchronized activity. In addition, spontaneous reductions in Ca(2+) were observed that inhibited Ca(2+) waves. N omega-nitro-L-arginine (100 micromol/L; nitric oxide antagonist) blocked the reductions in Ca(2+) and increased the frequency (approximately 19/min) of intracellular Ca(2+) waves within ICC-MY., Conclusions: ICC-SMs form a tightly coupled network that is able to generate and propagate slow waves. In contrast, Ca(2+) transients in ICC-MYs, which are normally not synchronized, have a similar duration and frequency as MPOs. Like MPOs, their activity is inhibited by nitrergic nerves and synchronized by excitatory nerves.

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., Methods: Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa., Results: Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation., Conclusions: The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca(2+) imaging techniques. Full colons were 158.1 +/- 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s(-1)). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca(2+) waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechanosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Background & Aims: Transit of fecal material through the human colon takes > or =30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena., Methods: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca(2+) imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit., Results: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-omega-nitro-L-arginine (100 micromol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca(2+) imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS +ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N-omega-nitro-L-arginine (100 micromol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility., Conclusions: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS +ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.

In mammals that develop rectal aganglionosis, the aganglionic segment still exhibits spontaneous phasic contractions that contribute to dysmotility and pseudoobstruction in this region. However, almost nothing is known about the mechanisms that generate these myogenic contractions or the effects of aganglionosis on the generation of Ca(2+) waves that underlie contractions of the longitudinal muscle (LM) and circular muscle (CM). In a mouse model of Hirschsprung's disease [endothelin type B receptor-deficient (Ednrb(s-l)/Ednrb(s-l)) mice], the Ca(2+) indicator fluo-4 was used to simultaneously monitor the temporal activation and spread of intercellular Ca(2+) waves in the LM and CM during spontaneous colonic motor activities. During the intervals between colonic migrating motor complexes (CMMCs) in control mice, Ca(2+) waves discharged asynchronously between the LM and CM. However, in these same mice, during CMMCs, a burst of discreet Ca(2+) waves fired simultaneously in both muscle layers, where the propagation velocity of Ca(2+) waves significantly increased, as did the rate of initiation and number of collisions between Ca(2+) waves. Hexamethonium (300 microM) or atropine (1 microM) prevented synchronized firing of Ca(2+) waves. In the aganglionic distal colon of Ednrb(s-l)/Ednrb(s-l) mice, not only were CMMCs absent, but Ca(2+) waves between the two muscle layers fired asynchronously, despite increased propagation velocity. The generation of CMMCs in control mice involves synchronized firing of enteric motor nerves to both the LM and CM, explaining the synchronized firing of discreet Ca(2+) waves between the two muscle layers. Aganglionosis results in a sporadic and sustained asynchrony in Ca(2+) wave firing between the LM and CM and an absence of CMMCs.

DiI (1,1'didodecyl-3,3,3',3'-tetramethylindocarbecyanine perchlorate) retrograde labelling and intracellular electrophysiological techniques were used to investigate the mechanisms underlying the generation of spontaneously occurring colonic migrating myoelectric complexes (colonic MMCs) in mice. In isolated, intact, whole colonic preparations, simultaneous intracellular electrical recordings were made from pairs of circular muscle (CM) cells during colonic MMC activity in the presence of nifedipine (1-2 microm). During the intervals between colonic MMCs, spontaneous inhibitory junction potentials (IJPs) were always present. The amplitudes of spontaneous IJPs were highly variable (range 1-20 mV) and occurred asynchronously in the two CM cells, when separated by 1 mm in the longitudinal axis. Colonic MMCs occurred every 151 +/- 7 s in the CM and consisted of a repetitive discharge of cholinergic rapid oscillations in membrane potential (range: 1-20 mV) that were superimposed on a slow membrane depolarization (mean amplitude: 9.6 +/- 0.5 mV; half-duration: 25.9 +/- 0.7 s). During the rising (depolarizing) phase of each colonic MMC, cholinergic rapid oscillations occurred simultaneously in both CM cells, even when the two electrodes were separated by up to 15 mm along the longitudinal axis of the colon. Smaller amplitude oscillations (< 5 mV) showed poor temporal correlation between two CM cells, even at short electrode separation distances (i.e. < 1 mm in the longitudinal axis). When the two electrodes were separated by 20 mm, all cholinergic rapid oscillations and IJPs in the CM (regardless of amplitude) were rarely, if ever, coordinated in time during the colonic MMC. Cholinergic rapid oscillations were blocked by atropine (1 microm) or tetrodotoxin (1 microm). Slow waves were never recorded from any CM cells. DiI labelling showed that the maximum projection length of CM motor neurones and interneurones along the bowel was 2.8 mm and 13 mm, respectively. When recordings were made adjacent to either oral or anal cut ends of the colon, the inhibitory or excitatory phases of the colonic MMC were absent, respectively. In summary, during the colonic MMC, cholinergic rapid oscillations of similar amplitudes occur simultaneously in two CM cells separated by large distances (up to 15 mm). As this distance was found to be far greater than the projection length of any single CM motor neurone, we suggest that the generation of each discrete cholinergic rapid oscillation represents a discreet cholinergic excitatory junction potential (EJP) that involves the synaptic activation of many cholinergic motor neurones simultaneously, by synchronous firing in many myenteric interneurones. Our data also suggest that ascending excitatory and descending inhibitory nerve pathways interact and reinforce each other.

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells (<200 microm apart) at the oral region of the colon showed that excitatory junction potentials (EJPs) discharged synchronously in both muscle layers for periods of up to 6 h. Similarly, at the anal region of the colon, inhibitory junction potentials (IJPs) discharged synchronously in the two muscle layers. Quadruple recordings from LM and CM orally at the same time as from the LM and CM anally revealed that IJPs occurred synchronously in the LM and CM anally at the same time as EJPs in LM and CM located 20 mm orally. Oral EJPs and anal IJPs were linearly related in amplitude between the two muscle layers. Spatiotemporal maps generated from simultaneous video imaging of the movements of the colon, combined with intracellular recordings, revealed that some LM contractions orally could be correlated in time with IJPs in CM cells anally. N(omega)-nitro-L-arginine (L-NA; 100 microM) abolished the IJP in LM, whereas a prominent L-NA-resistant "fast" IJP was always observed in CM. In summary, in stretched preparations, synchronized EJPs in both LM and CM orally are generated by synchronized firing of many ascending interneurons, which simultaneously activate excitatory motor neurons to both muscle layers. Similarly, synchronized IJPs in both LM and CM anally are generated by synchronized firing of many descending interneurons, which simultaneously activate inhibitory motor neurons to both muscle layers. This synchronized motor activity ensures that both muscles around the entire circumference are excited orally at the same time as inhibited anally, thus producing net aboral propulsion.

Simultaneous intracellular recordings were made from pairs of circular muscle (CM) cells, at the oral and anal ends of a segment of guinea-pig distal colon, to investigate the neuronal mechanisms underlying faecal pellet propulsion. When a minimum degree of circumferential stretch was applied to sheet preparations of colon, recordings from CM cells revealed either no ongoing junction potentials, or alternatively, small potentials usually < 5 mV in amplitude. Maintained circumferential stretch applied to these preparations evoked an ongoing discharge of excitatory junction potentials (EJPs) at the oral recording site (range: 1-25 mV), which lasted for up to 6 h. The onset of each large oral EJP was time-locked with the onset of an inhibitory junction potential (IJP) at an anal recording electrode, located 2 cm from the oral recording. Similar results were obtained in isolated intact tube preparations of colon, when recordings were made immediately oral and anal of an artificial faecal pellet. The amplitudes of many large (> 5 mV) oral EJPs were linearly related to the amplitudes of anal IJPs occurring 20 mm apart. In the absence of an L-type Ca(2+) channel blocker, action potentials occurred on each large oral EJP. Synchronized discharges of stretch-activated EJPs and IJPs were preserved following pretreatment with capsaicin (10 microM), were unaffected by nifedipine (1 microM) and did not require the mucosa or submucous plexus. EJPs and IJPs were abolished by hexamethonium (300 microM) or tetrodotoxin (1 microM), but persisted in the presence of pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 microM) or an NK(3) tachykinin receptor antagonist (Neurokinin A 4-10; 100 nM to 5 microM). In summary, maintained circumferential stretch of the distal colon activates a population of intrinsic mechanosensory neurons that generate repetitive firing of ascending excitatory and descending inhibitory pathways to CM. These mechanosensory neurons, which may be interneurons, are stretch sensitive, rather than muscle tension sensitive, since they are resistant to muscular paralysis. We suggest the synchrony in onset of oral EJPs and anal IJPs over large regions of colon is due to synchronous synaptic activation of ascending and descending interneurons.

Ca2+ wave activity was monitored in the longitudinal (LM) layer of isolated murine caecum and proximal colon at 35 degrees C with fluo-4 AM and an iCCD camera. Both intracellular (within LM cells) and intercellular (also spreading from cell to cell) Ca2+ waves were observed. Intracellular Ca2+ waves were associated with a lack of muscle movement whereas intercellular Ca2+ waves, which were five times more intense than intracellular waves, were often associated with localized contractions. Several intracellular Ca2+ waves were present at the same time in individual LM cells. Waves in adjacent LM cells were not coordinated and were unaffected by TTX (1 microM) but were blocked by IP3 receptor antagonists xestospongin-C (Xe-C; 2 microM) or 2-aminoethyl diphenylborate (2-APB; 25 microM), and by ryanodine (10 microM). Caffeine (5 mM) restored wave activity following blockade with Xe-C. NiCl2 (1 mM) blocked intracellular Ca2+ waves, and nicardipine (2 microM) reduced their frequency and intensity, but did not affect their velocity, suggesting the sarcoplasmic reticulum may be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves often occurred in bursts and propagated rapidly across sizeable regions of the LM layer and were blocked by heptanol (0.5 mM). Intercellular Ca2+ waves were dependent upon neural activity, external Ca2+ entry through L-type Ca2+ channels, and amplification via calcium-induced calcium release (CICR). In conclusion, intracellular Ca2+ waves, which may reduce muscle excitability, are confined to individual LM cells. They depend upon Ca2+ release from internal Ca2+ stores and are likely to be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves, which are likely to underlie smooth muscle tone, mixing and propulsion, depend upon neural activity, muscle action potential propagation and amplification by CICR.

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 microm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 microM) or tetrodotoxin (1 microM) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 microM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca(2+) channels.

1. In isolated, stretched, flat-sheet preparations of guinea-pig distal colon, simultaneous intracellular recordings were made from pairs of circular muscle (CM) cells to map the region of smooth muscle at which spontaneous junction potentials (sJPs) were coordinated in both space and time. 2. Spontaneous inhibitory junction potentials (sIJPs) and excitatory junction potentials (sEJPs) were recorded from all animals with varying frequencies and amplitudes (up to 25 mV). 3. Large amplitude (> or = 9 mV) sIJPs or sEJPs with near-identical amplitudes and time courses were recorded synchronously from two CM cells, even when the two electrodes were separated by up to 11 mm in the circumferential axis and < or = 4 mm in the longitudinal axis. However, smaller (< 9 mV) sIJPs or sEJPs were less coordinated and exhibited greater variability in their times to peak. 4. The standard deviation (S.D.) for the time difference between the peaks of sJPs was related to the amplitude of the events and the distance between the electrodes. The S.D. for large amplitude JPs (approximately 30 ms), which was less than that for small JPs (approximately 150 ms), remained constant across the circumferential axis (at least up to 6 mm), but declined rapidly for distances > or = 2 mm in the longitudinal axis. 5. Current injection (up to 8 nA) into a single CM cell elicited electrotonic potentials in neighbouring CM cells, only when the two electrodes were separated by less than 100 microm circumferentially. Beyond 50 microm electronic potentials were rarely detected. 6. Tetrodotoxin (TTX; 1 microM) abolished all sJPs, whereas hexamethonium (300 microM) either abolished, or substantially reduced all sJPs. 7. Nitro-L-arginine (L-NA; 100 microM) abolished the slow repolarisation phase of sIJPs without any apparent effect on the amplitude of sIJPs. Apamin abolished the fast, initial component of sIJPs, suggesting synchronous release of two inhibitory neurotransmitters during the sIJP. Atropine (1 microM) abolished sEJPs. 8. No sJPs were recorded from the CM layer when it was separated from the myenteric plexus. 9. In conclusion, sIJPs and sEJPs in colonic CM occur synchronously over large regions of the smooth muscle syncitium. The results are discussed in relation to the idea that spontaneous junction potentials in colonic CM are not monoquantal events, but are generated by the simultaneous release of transmitter from many release sites, due to the synchronous activation of many enteric motor neurons.