Guinea Pig Distal Colon Research Articles

Stimulation of extrinsic sympathetic nerves differentially affects neurogenic motor activity in guinea pig distal colon.

The speed of pellet propulsion through the isolated guinea pig distal colon in vitro significantly exceeds in vivo measurements, suggesting a role for inhibitory mechanisms from sources outside the gut. The aim of this study was to investigate the effects of sympathetic nerve stimulation on three different neurogenic motor behaviors of the distal colon: transient neural events (TNEs), colonic motor complexes (CMCs), and pellet propulsion. To do this, segments of guinea pig distal colon with intact connections to the inferior mesenteric ganglion (IMG) were set up in organ baths allowing for simultaneous extracellular suction electrode recordings from smooth muscle, video recordings for diameter mapping, and intraluminal manometry. Electrical stimulation (1-20 Hz) of colonic nerves surrounding the inferior mesenteric artery caused a statistically significant, frequency-dependent inhibition of TNEs, as well as single pellet propulsion, from frequencies of 5Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10Hz and greater. Phentolamine (3.6μM) abolished effects of colonic nerve stimulation, consistent with a sympathetic noradrenergic mechanism. Sympathetic inhibition was constrained to regions with intact extrinsic nerve pathways, allowing normal motor behaviors to continue without modulation in adjacent extrinsically denervated regions of the same colonic segments. The results demonstrate differential sensitivities to sympathetic input among distinct neurogenic motor behaviors of the colon. Together with findings indicating CMCs activate colo-colonic sympathetic reflexes through the IMG, these results raise the possibility that CMCs may paradoxically facilitate suppression of pellet movement in vivo, through peripheral sympathetic reflex circuits.

Mechanisms underlying initiation of propulsion in guinea pig distal colon.

Colonic motor complexes (CMCs) are a major neurogenic activity in guineapig distal colon. The identity of the enteric neurons that initiate this activity is not established. Specialized intrinsic primary afferent neurons (IPANs) are a major candidate. We aimed to test this hypothesis. To do this, segments of guineapig distal colon were suspended vertically in heated organ baths and propulsive forces acting on a pellet inside the lumen were recorded by isometric force transducer while pharmacological agents were applied to affect IPAN function. In the absence of drugs, CMCs acted periodically on the pellet, generating peak propulsive forces of 12.7 ± 5 g at 0.56 ± 0.22 cpm, lasting 49 ± 17 s (215 preparations; n = 60). Most but not all CMCs were abolished by nicotinic receptor blockade to inhibit fast excitatory synaptic transmission (50/62 preparations; n = 25). Remarkably, CMCs inhibited by hexamethonium were restored by a pharmacological strategy that aimed to enhance IPAN excitability. Thus, CMCs were restored by increased smooth muscle tension (using BAY K8644, bethanechol or carbachol) and by IPAN excitation using phorbol dibutyrate; NK3 receptor agonist, senktide; and partially by αCGRP. The IPAN inhibitor, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (DCEBIO), decreased CMC frequency. CGRP, but not NK3-receptor antagonists, decreased CMC frequency in naive preparations. Finally, CMCs were blocked by tetrodotoxin, and this was not reversed by any drugs listed above. These results support a major role for IPANs that does not require fast synaptic transmission, in the periodic initiation of neurogenic propulsive contractions. Endogenous CGRP plays a role in determining CMC frequency, whereas further unidentified signaling pathways may determine their amplitude and duration.NEW & NOTEWORTHY The colonic motor complex (CMC) initiates propulsion in guinea pig colon. Here, CMCs evoked by an intraluminal pellet were restored during nicotinic receptor blockade by pharmacological agents that directly or indirectly enhance intrinsic primary afferent neuron (IPAN) excitability. IPANs are the only enteric neuron in colon that contain CGRP. Blocking CGRP receptors decreased CMC frequency, implicating their role in CMC initiation. The results support a role for IPANs in the initiation of CMCs.

Roles of three distinct neurogenic motor patterns during pellet propulsion in guinea-pig distal colon.

Enteric neural circuits enable isolated preparations of guinea-pig distal colon to propel solid and fluid contents by a self-sustaining neuromechanical loop process. In addition there are at least three neural mechanisms which are not directly involved in propulsion: cyclic motor complexes, transient neural events and distal colon migrating motor complexes. In excised guinea-pig colon we simultaneously recorded high resolution manometry, video-imaging of colonic wall movements and electrophysiological recordings from smooth muscle, which enabled us to identify mechanisms that underlie the propulsion of colonic content. The results show that the intermittent propulsion during emptying of the multiple natural faecal pellets is due to the intermittent activation of cyclic motor complexes and this is facilitated by transient neural events. Loss or dysfunction of these activities is likely to underlie disordered gastrointestinal transit. It is well known that there are different patterns of electrical activity in smooth muscle cells along different regions of the gastrointestinal tract. These different patterns can be generated by myogenic and/or neurogenic mechanisms. However, what patterns of electrical activity underlie the propulsion of natural faecal content remains unknown, particularly along the large intestine, where large quantities of water are reabsorbed and semi-solid faeces form. In this study, we developed a novel approach which enables for the first time the simultaneous recording of high resolution intraluminal manometry, electrophysiology from the smooth muscle, and spatio-temporal video imaging of colonic wall movements. Using this approach we were able to reveal the nature of enteric neuromuscular transmission and patterns of motor activity responsible for the movement of content. Three distinct neurogenic patterns of electrical activity were recorded even in the absence of propulsive movement. These were the cyclic motor complexes (CMCs), the transient neural events (TNEs) and the slowly propagating distal colonic migrating motor complexes (DCMMCs). We present evidence that the initiation of pellet propulsion is due to a cyclic motor complex (CMC) occurring oral to the pellet. Furthermore, we discovered that the intermittent propulsion of natural faecal pellets is generated by intermittent activation of CMCs; and this propulsion is facilitated by hexamethonium-sensitive TNEs. However, TNEs were not required for propulsion. The findings reveal the patterns of electrical activity that underlie propulsion of natural colonic content and demonstrate that propulsion is generated by a complex interplay between distinct enteric neural circuits.

Identification of multiple distinct neurogenic motor patterns that can occur simultaneously in the guinea pig distal colon

In the guinea pig distal colon, nonpropulsive neurally mediated motor patterns have been observed in different experimental conditions. Isolated segments of guinea pig distal colon were used to investigate these neural mechanisms by simultaneously recording wall motion, intraluminal pressure, and smooth muscle electrical activity in different conditions of constant distension and in response to pharmacological agents. Three distinct neurally dependent motor patterns were identified: transient neural events (TNEs), cyclic motor complexes (CMC), and distal colon migrating motor complexes (DCMMC). These could occur simultaneously and were distinguished by their electrophysiological, mechanical, and pharmacological features. TNEs occurred at irregular intervals of ~3s, with bursts of action potentials at 9 Hz. They propagated orally at 12 cm/s via assemblies of ascending cholinergic interneurons that activated final excitatory and inhibitory motor neurons, apparently without involvement of stretch-sensitive intrinsic primary afferent neurons. CMCs occurred during maintained distension and consisted of clusters of closely spaced TNEs, which fused to cause high-frequency action potential firing at 7 Hz lasting ~10 s. They generated periodic pressure peaks mediated by stretch-sensitive intrinsic primary afferent neurons and by cholinergic interneurons. DCMMCs were generated by ongoing activity in excitatory motor neurons without apparent involvement of stretch-sensitive neurons, cholinergic interneurons, or inhibitory motor neurons. In conclusion, we have identified three distinct motor patterns that can occur concurrently in the isolated guinea pig distal colon. The mechanisms underlying the generation of these neural patterns likely involve recruitment of different populations of enteric neurons with distinct temporal activation properties.

Morphological evidence for novel enteric neuronal circuitry in guinea pig distal colon.

The gastrointestinal (GI) tract is unique compared to all other internal organs; it is the only organ with its own nervous system and its own population of intrinsic sensory neurons, known as intrinsic primary afferent neurons (IPANs). How these IPANs form neuronal circuits with other functional classes of neurons in the enteric nervous system (ENS) is incompletely understood. We used a combination of light microscopy, immunohistochemistry and confocal microscopy to examine the topographical distribution of specific classes of neurons in the myenteric plexus of guinea-pig colon, including putative IPANs, with other classes of enteric neurons. These findings were based on immunoreactivity to the neuronal markers, calbindin, calretinin and nitric oxide synthase. We then correlated the varicose outputs formed by putative IPANs with subclasses of excitatory interneurons and motor neurons. We revealed that calbindin-immunoreactive varicosities form specialized structures resembling 'baskets' within the majority of myenteric ganglia, which were arranged in clusters around calretinin-immunoreactive neurons. These calbindin baskets directly arose from projections of putative IPANs and represent morphological evidence of preferential input from sensory neurons directly to a select group of calretinin neurons. Our findings uncovered that these neurons are likely to be ascending excitatory interneurons and excitatory motor neurons. Our study reveals for the first time in the colon, a novel enteric neural circuit, whereby calbindin-immunoreactive putative sensory neurons form specialized varicose structures that likely direct synaptic outputs to excitatory interneurons and motor neurons. This circuit likely forms the basis of polarized neuronal pathways underlying motility.

The Effects of Short Chain Fatty Acid Mixtures on Colonic Motility

The effects of nutrients on a variety of colonic function has been well studied. Recent evidence has suggested that sensing of many different intraluminal nutrients (glucose, amino acids) is accomplished through plasma membrane receptors within the intestinal epithelium. These membrane receptors are often linked to hormone release from enteroendocrine cells. While many digested nutrients are present at higher intraluminal concentrations within the small intestine before they are absorbed, short chain fatty acids (SCFAs) in particular are generated by bacterial metabolism of nutrients within the colon. Based on previous studies, the most prominent individual SCFAs (butyrate, acetate, and propionate) have differential effects on cellular signaling and motility within the colon. However, the overall effects of mixtures of these SCFAs, which are more similar to intraluminal conditions, have not been elucidated. In this study, we measured colonic motility of both the proximal and distal colon in response to SCFA mixtures. Motility in the proximal colon of guinea pigs was measured as full‐propagating, shortpropagating, or non‐propagating contractions through video capture and subsequent generation of spatiotemporal maps. Motility in guinea pig distal colon was measured by velocity of fecal pellet propulsion. An 30mM SCFA mixture with a molar ratio of 3:2:1 (acetate: butyrate: propionate) increased velocity of fecal pellet propulsion. This increase in distal colonic motility was inhibited by GR113808, an antagonist of 5‐HT4 receptors, suggesting that mediation of motility effects was through serotonin. Statistical comparisons of pellet velocity were made by one‐way repeated measures ANOVA. In the proximal colon, the same SCFA mixture reduced the presence of short‐propagating contractions (<50% of the tissue length) while not affecting full‐propagating contractions. These results suggest that SCFA mixtures have differential effects on propulsive motility in the proximal and distal colon of guinea pigs. Future work will elucidate the presence of the FFAR2 and FFAR3 receptors in relation to enteroendocrine cells containing serotonin and other endocrine mediators of colonic function (peptide YY, neurotensin).Support or Funding InformationThis work was supported by a summer research grant to DMK from Loyola University Maryland and a small grant by the VCU postdoctoral association. DMK was also partially supported by an NIGMS IRACDA grant GM093857 to VCU.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The novel, potent and highly selective 5-HT4 receptor agonist YH12852 significantly improves both upper and lower gastrointestinal motility.

5-HT4 receptor agonists have been shown to be effective at treating various gastrointestinal tract disorders. However, a lack of selectivity against off-targets has been a limiting factor for their clinical use. The binding affinity and selectivity of YH12852 for human 5-HT4(a) receptor in CHO-K1 cells were evaluated using radioligand binding assays, and agonistic activity was assessed using a β-lactamase reporter system. Contractile activity and propulsive motility were measured in the guinea pig isolated distal colon. Its prokinetic effect on the gastrointestinal tract was evaluated in guinea pigs, dogs and monkeys. Its tissue distribution was evaluated in rats. YH12852 exhibited high affinity and potency for human recombinant 5-HT4(a) receptor with high selectivity over other 5-HT and non-5-HT receptors, ion channels, enzymes and transporters. YH12852 induced contractions and increased propulsive motility in guinea pig isolated colon. These effects were abolished by the 5-HT4 receptor antagonist GR113808. YH12852 increased defecation more effectively than prucalopride in guinea pigs and dogs and improved gastric emptying more effectively than mosapride in guinea pigs, dogs and monkeys. YH12852 was highly distributed to the gastrointestinal tract as the target organ. The high in vitro potency and selectivity of YH12852 for 5-HT4 receptor translated into potent in vivo efficacy with good tolerability. YH12852 significantly improved both upper and lower bowel motility in the animal models tested and has the potential to address considerable unmet needs in patients with functional constipation, gastroparesis or both.

New insights into neurogenic cyclic motor activity in the isolated guinea-pig colon.

The contents of the guinea pig distal colon consist of multiple pellets that move anally in a coordinated manner. This row of pellets results in continued distention of the colon. In this study, we have investigated quantitatively the features of the neurally dependent colonic motor patterns that are evoked by constant distension of the full length of guinea-pig colon. Constant distension was applied to the excised guinea-pig by high-resolution manometry catheters or by a series of hooks. Constant distension elicited regular Cyclic Motor Complexes (CMCs) that originated at multiple different sites along the colon and propagated in an oral or anal direction extending distances of 18.3±10.3cm. CMCs were blocked by tetrodotoxin (TTX; 0.6μmolL-1 ), hexamethonium (100μmolL-1 ) or hyoscine (1μmolL-1 ). Application of TTX in a localized compartment or cutting the gut circumferentially disrupted the spatialcontinuity of CMCs. Localized smooth muscle contraction was not required for CMC propagation. Shortening the length of the preparations or disruption of circumferential pathways reduced the integrity and continuity of CMCs. CMCs are a distinctive neurally dependent cyclic motor pattern, that emerge with distension over long lengths of the distal colon. They do not require changes in muscle tension or contractility to entrain the neural activity underlying CMC propagation. CMCs are likely to play an important role interacting with the neuromechanical processes that time the propulsion of multiple natural pellets and may be particularly relevant in conditionsof impaction or obstruction, where long segments of colon are simultaneously distended.

Measurement of strains experienced by viscerofugal nerve cell bodies during mechanosensitive firing using digital image correlation.

Mechanosensory neurons detect physical events in the local environments of the tissues that they innervate. Studies of mechanosensitivity of neurons or nerve endings in the gut have related their firing to strain, wall tension, or pressure. Digital image correlation (DIC) is a technique from materials engineering that can be adapted to measure the local physical environments of afferent neurons at high resolution. Flat-sheet preparations of guinea pig distal colon were set up with arrays of tissue markers in vitro. Firing of single viscerofugal neurons was identified in extracellular colonic nerve recordings. The locations of viscerofugal nerve cell bodies were inferred by mapping firing responses to focal application of the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide. Mechanosensory firing was recorded during load-evoked uniaxial or biaxial distensions. Distension caused movement of surface markers which was captured by video imaging. DIC tracked the markers, interpolating the mechanical state of the gut at the location of the viscerofugal nerve cell body. This technique revealed heterogeneous load-evoked strain within preparations. Local strains at viscerofugal nerve cell bodies were usually smaller than global strain measurements and correlated more closely with mechanosensitive firing. Both circumferential and longitudinal strain activated viscerofugal neurons. Simultaneous loading in circumferential and longitudinal axes caused the highest levels of viscerofugal neuron firing. Multiaxial strains, reflecting tissue shearing and changing area, linearly correlated with mechanosensory firing of viscerofugal neurons. Viscerofugal neurons were mechanically sensitive to both local circumferential and local longitudinal gut strain, and appear to lack directionality in their stretch sensitivity.

Disruption of Serotonin Signaling Results in Loss of Coordinated Tone Development and Subsequent Inhibition of Peristalsis in Guinea Pig Distal Colon

This project was designed to examine the effects of the selective serotonin reuptake inhibitor, Fluoxetine, on colonic motility using the isolated guinea pig distal colon. The Gastrointestinal Motility Monitor system (“GIMM”, Catamount Research and Development Inc., St. Albans, VT) was used to analyze peristaltic motility during fecal pellet propulsion, as well as to conduct spatiotemporal analysis of motility patterns. Peristaltic motility in colonic segments was significantly slowed by Fluoxetine at concentrations of 20, 30 and 40 μM (p<0.05 vs. vehicle). Indeed, in colonic segments treated with 40 μM Fluoxetine, peristaltic motility was completely abolished (at least 1 of 3 trials with no peristaltic motility) in 4 out of 5 experimental preparations. This striking disruption in peristaltic motility by Fluoxetine was associated with a pronounced change in spatiotemporal motility patterns. In the presence of 40 μM Fluoxetine, average colonic diameter during the motility trial was substantially increased relative to pre‐treatment diameter (36.6 ± 21.5 % increase from baseline diameter, n=5). In vehicle‐treated colonic segments, average diameter remained relatively unchanged from pre‐treatment levels (0.5 ± 3.5 % change from baseline diameter, n=11). These findings suggest that serotonin signaling in the wall of the gut is required for normal peristaltic propulsive motility, and that disruption of serotonin signaling using Fluoxetine results in a loss of coordinated tone in the wall of the gut. Understanding the exact role of serotonin in normal gut motility is essential to gaining insights into novel therapeutic strategies for treating bowel dysfunction.Support or Funding InformationThis study was supported by Catamount Research and Development

Su1593 Cyclic Neurogenic Motor Patterns in the Guinea-Pig Distal Colon

Su1593 Cyclic Neurogenic Motor Patterns in the Guinea-Pig Distal Colon

Abundant 5‐HT Release from EC Cells Disrupts Colonic Motility

Serotonin (5‐HT) that is synthesized by enterochromaffin (EC) cells in the intestinal mucosa can activate the ascending contractile and descending relaxant limbs of the peristaltic reflex. However, recent studies have demonstrated that peristalsis persists in the absence of mucosal 5‐HT, raising the question of whether mucosal 5‐HT actually influences propulsive motility. In this study we evaluated the actions of emetic drugs (ipecac, emetine and cisplatin), which are known to induce massive release of 5‐HT from EC cells, on propulsive motility in isolated segments of guinea pig distal colon. A Gastrointestinal Motility Monitor (GIMM; Catamount Research) system was used to record and analyze the rate of fecal pellet propulsive motility. Following a 30 minute equilibration period, pellet velocity was evaluated over three trials separated by 5 minute rest periods, and under control conditions was typically in the range of 1.8‐2.4 mm/sec. Drugs were then added to the solution for 10 min, and an additional 3 trials were recorded. Intraluminal infusion of ipecac (2‐10%), emetine (1‐10mM) or cisplatin (3µM‐1mM) to the bath slowed motility in a concentration‐dependent manner, and halted transit at high concentrations. Bath application of the 5HT3/5HT4 antagonist SDZ‐205‐557 hydrochloride (10µM) inhibited the effects of the emetic compounds, but intraluminal application of the antagonist had no effect. In summary, extensive activation of 5‐HT release from EC cells over an entire segment of the colon has a negative impact on propulsive motility. These findings support the concept that release of mucosal 5‐HT can influence propulsive motility in the intestines. Supported by DK62267

Neurotensin Partially Mediates the Monosodium Glutamate (MSG)‐Induced Peristaltic Reflex

Activation of nutrient sensors throughout the gastrointestinal (GI) tract can affect both motility and secretory functions. Our recent studies have shown that monosodium glutamate (MSG) and the amino acid L-Cysteine can activate the nutrient sensing T1R1/T1R3 umami receptor and increase propulsive motility in the distal colon. Other groups have shown that activation of these receptors in the upper GI tract can lead to release of hormone and paracrine mediators from enteroendocrine cells (EECs). In this study we investigated the mechanism of MSG-induced increases in propulsive motility through the release of the paracrine mediator neurotensin (NT). We investigated MSG-induced release of NT in both a mouse enteroendocrine cell line, STC-1, and rat colon flat sheet preparation. In both STC-1 cells and guinea pig distal colon tissue T1R1/T1R3 receptors were co-localized to cells containing NT as assessed by immunohistochemistry. MSG induced NT release in both STC-1 cells and mucosal flat sheet preparations from rat colon. The NT release by MSG in rat colon was augmented by the addition of inosine-5'-monophosphate (IMP) suggesting that the effect was downstream of T1R1/T1R3 activation. MSG induced both the ascending contraction and descending relaxation components of the peristaltic reflex in rat colon, which was reduced by the addition of the NT1 receptor antagonist SR48692. The increased pellet propulsion velocity induced by MSG (132% of control) was reduced by the addition of the pan-NT receptor inhibitor SR142498 (115% of control). These results suggest that MSG-induced propulsive motility is partially mediated by the release of NT from EECs within the colonic mucosa. This research was supported by NIDDK grant DK034153 (JRG) and NIGMS grant GM093857.

Neurochemical characterization of extrinsic nerves in myenteric ganglia of the guinea pig distal colon.

Extrinsic nerves to the gut influence the absorption of water and electrolytes and expulsion of waste contents, largely via regulation of enteric neural circuits; they also contribute to control of blood flow. The distal colon is innervated by extrinsic sympathetic and parasympathetic efferent and spinal afferent neurons, via axons in colonic nerve trunks. In the present study, biotinamide tracing of colonic nerves was combined with immunohistochemical labeling for markers of sympathetic, parasympathetic, and spinal afferent neurons to quantify their relative contribution to the extrinsic innervation. Calcitonin gene-related peptide, vesicular acetylcholine transporter, and tyrosine hydroxylase, which selectively label spinal afferent, parasympathetic, and sympathetic axons, respectively, were detected immunohistochemically in 1 ± 0.5% (n = 7), 15 ± 4.7% (n = 6), and 24 ± 4% (n = 7) of biotinamide-labeled extrinsic axons in myenteric ganglia. Immunoreactivity for vasoactive intestinal polypeptide, nitric oxide synthase, somatostatin, and vesicular glutamate transporters 1 and 2 accounted for a combined maximum of 14% of biotinamide-labeled axons in myenteric ganglia. Thus, a maximum of 53% of biotinamide-labeled extrinsic axons in myenteric ganglia were labeled by antisera to one of these eight markers. Viscerofugal neurons were also labeled by biotinamide. They had distinct morphologies and spatial distributions that correlated closely with their immunoreactivity for nitric oxide synthase and choline acetyltransferase. As reported for the rectum, nearly half of all extrinsic nerve fibers to the distal colon lack the key immunohistochemical markers commonly used for their identification. Their abundance may therefore have been significantly underestimated in previous immunohistochemical studies.

Activation of the umami taste receptor (T1R1/T1R3) initiates the peristaltic reflex and pellet propulsion in the distal colon.

Intraluminal nutrients in the gut affect the peristaltic reflex, although the mechanism is not well defined. Recent evidence supports the presence of taste receptors and their signaling components in enteroendocrine cells, although their function is unclear. This study aimed to determine if nutrients modify colonic motility through activation of taste receptors. Colonic sections were immunostained for the umami taste receptor T1R1/T1R3, which mediates the response to umami ligands, such as monosodium glutamate (MSG), in taste cells. Ascending contraction, descending relaxation, and calcitonin gene-related peptide release were measured in three-chamber flat-sheet preparations of rat colon in response to MSG alone or with inosine 5'-monophosphate (IMP). Velocity of artificial fecal pellet propulsion was measured by video recording in guinea pig distal colon. T1R1/T1R3 receptors were present in enteroendocrine cells of colonic sections from human, rat, mouse, and guinea pig. MSG initiated ascending contraction and descending relaxation components of the peristaltic reflex and calcitonin gene-related peptide release in flat-sheet preparations. IMP augmented the MSG-induced effects, suggesting activation of T1R1/T1R3 receptors. In T1R1(-/-) mice, mucosal stroking, but not MSG, elicited a peristaltic reflex. Intraluminal perfusion of MSG enhanced the velocity of artificial fecal pellet propulsion, which was also augmented by IMP. Propulsion was also increased by l-cysteine, but not l-tryptophan, supporting a role of T1R1/T1R3 receptors. We conclude that T1R1/T1R3 activation by luminal MSG or l-cysteine elicits a peristaltic reflex and CGRP release and increases the velocity of pellet propulsion in distal colon. This mechanism may explain how nutrients regulate colonic propulsion.

Evidence for tachykinin NK3 receptors-triggered peptide YY release from isolated guinea-pig distal colon

The anorectic gut hormone, peptide YY (PYY), is released from colonic mucosal endocrine cells, but little is known about the role for tachykinin NK3 receptor in the control of PYY release from the colonic mucosa. We investigated the functional role for NK3 receptors in the control of PYY release from isolated guinea-pig distal colon, and the role for NK3 receptors-triggered PYY release in the control of colonic motility. Isolated colonic preparations were mounted in organ baths for measurement of PYY release and mechanical activity. The release of PYY from these preparations was determined by enzyme immunoassays. The NK3 receptor agonist senktide produced a tetrodotoxin/atropine-sensitive sustained increase in the release of PYY from the colonic preparations. Basal PYY release was transiently inhibited by the NK3 receptor antagonist SB222200. The neuropeptide Y1 receptor antagonist BIBO3304 produced a leftward shift of the concentration–response curves for senktide-evoked neurogenic contraction, but neither the neuropeptide Y2 receptor antagonist BIIE0246 nor the neuropeptide Y5 receptor antagonist CGP71683 affected the senktide concentration–response curves. NK3 receptors appear to play an important role in the control of PYY release from colonic mucosa, and NK3 receptor-triggered PYY release can exert Y1 receptor-mediated inhibition of tachykinergic neuromuscular transmission. This indicates a pathophysiological role for the NK3 receptor-triggered PYY release in the control of colonic motility.

Targeted electrophysiological analysis of viscerofugal neurons in the myenteric plexus of guinea-pig colon

Enteric viscerofugal neurons are mechanosensory interneurons that form the afferent limb of intestino-intestinal reflexes involving prevertebral sympathetic neurons. Fast synaptic inputs to viscerofugal neurons arise from other enteric neurons, but their sources are unknown. We aimed to describe the origins of synaptic inputs to viscerofugal neurons by mapping the locations of their cell bodies within the myenteric plexus. Viscerofugal neuron somata were retrogradely traced with 1,1′-didodecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (DiI) from colonic nerve trunks and impaled with microelectrodes, in longitudinal muscle/myenteric plexus preparations of the guinea-pig distal colon (39 impalements, n=14). Thirty-eight viscerofugal neurons were uni-axonal and had the electrophysiological characteristics of myenteric S-neurons; one neuron was multipolar with AH-neuron electrophysiological characteristics. Depolarizing current pulses evoked either single- or multiple action potentials in viscerofugal neurons (range 1–25 spikes, 500ms, 100–900pA, 21 cells). Electrical stimulation of internodal strands circumferential to viscerofugal neurons evoked fast excitatory postsynaptic potentials (EPSPs) in 19/24 cells. Focal pressure-ejection of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10μm) directly onto viscerofugal nerve cell bodies evoked large depolarizations and action potentials (23±10mV, latency 350±230ms, 21/22 cells). DMPP was then focally applied to multiple sites, up to 3mm from the recorded viscerofugal neuron, to activate other myenteric S-neurons. In a few sites in myenteric ganglia, DMPP evoked repeatable fast EPSPs in viscerofugal neurons (latency 300±316ms, 38/394 sites, 10 cells). The cellular sources of synaptic inputs to viscerofugal neurons were located both orally and aborally (19 oral, 19 aboral), but the amplitude of oral inputs was consistently greater than aboral inputs (13.1±4.3mV vs. 10.1±4.8mV, respectively, p<0.05, paired t-test, n=6). Most impaled viscerofugal neurons were nitric oxide synthase (NOS) immunoreactive (20/27 cells tested). Thus, the synaptic connections onto viscerofugal neurons within the myenteric plexus suggest that multiple enteric neural pathways feed into intestino-intestinal reflexes, involving sympathetic prevertebral ganglia.

The velocity of pellet propulsion in guinea pig distal colon is differentially modified by short‐chain fatty acids (1110.8)

INTRODUCTION: Short‐chain fatty acids (SCFAs) are produced by commensurate bacteria in the colon. The type and concentration of SCFAs depend on dietary intake of carbohydrate and fiber. While most fermentation occurs in proximal colon, unabsorbed SCFAs entering the distal colon are likely to modify propulsion initiated by fecal pellet‐induced distension. AIM: To determine the effect of SCFAs on pellet propulsion in distal colon. METHODS: The distal colon was removed from guinea pig and placed in an organ bath containing Kreb’s buffer. Following equilibration, video tracking software was used to measure the velocity of propulsion of a clay pellet placed in the orad end of a segment. Pellets propulsion was repeated during intraluminal perfusion of Kreb’s buffer containing individual SCFAs at 30 mM caudad to the pellet at 0.1 ml/min. RESULTS: The mean velocity of pellet propulsion in the absence of SCFAs was 1.5 ± 0.2 mm/sec. Luminal perfusion with 30 mM sodium butyrate increased the velocity of pellet propulsion by 30 ± 4% (p&lt;0.05) whereas luminal perfusion with 30 mM sodium propionate decreased velocity of pellet propulsion by 75 ± 8%. Conclusion: Propionate had opposing effects to butyrate on the velocity of propulsion of artificial clay fecal pellets in the guinea pig distal colon. These studies suggest that the movement of feces in the colon depends not only on distension but also on the chemical (i.e. dietary) composition of the feces and the microbiota.Grant Funding Source: Supported by DK34153

Autonomic Nerve Regulation of Colonic Peristalsis in Guinea Pigs

Background/AimsColonic peristalsis is mainly regulated via intrinsic neurons in guinea pigs. However, autonomic regulation of colonic motility is poorly understood. We explored a guinea pig model for the study of extrinsic nerve effects on the distal colon.MethodsGuinea pigs were sacrificed, their distal colons isolated, preserving pelvic nerves (PN) and inferior mesenteric ganglia (IMG), and placed in a tissue bath. Fecal pellet propagation was conducted during PN and IMG stimulation at 10 Hz, 0.5 ms and 5 V. Distal colon was connected to a closed circuit system, and colonic motor responses were measured during PN and IMG stimulation.ResultsPN stimulation increased pellet velocity to 24.6 ± 0.7 mm/sec (n = 20), while IMG stimulation decreased it to 2.0 ± 0.2 mm/sec (n = 12), compared to controls (13.0 ± 0.7 mm/sec, P < 0.01). In closed circuit experiments, PN stimulation increased the intraluminal pressure, which was abolished by atropine (10−6 M) and hexamethonium (10−4 M). PN stimulation reduced the incidence of non-coordinated contractions induced by NG-nitro-L-arginine methyl ester (L-NAME; 10−4 M). IMG stimulation attenuated intraluminal pressure increase, which was partially reversed by alpha-2 adrenoceptor antagonist (yohimbine; 10−6 M).ConclusionsPN and IMG input determine speed of pellet progression and peristaltic reflex of the guinea pig distal colon. The stimulatory effects of PN involve nicotinic, muscarinic and nitrergic pathways. The inhibitory effects of IMG stimulation involve alpha-2 adrenoceptors.

Peristalsis and propulsion of colonic content can occur after blockade of major neuroneuronal and neuromuscular transmitters in isolated guinea pig colon

We recently identified hexamethonium-resistant peristalsis in the guinea pig colon. We showed that, following acute blockade of nicotinic receptors, peristalsis recovers, leading to normal propagation velocities of fecal pellets along the colon. This raises the fundamental question: what mechanisms underlie hexamethonium-resistant peristalsis? We investigated whether blockade of the major receptors that underlie excitatory neuromuscular transmission is required for hexamethonium-resistant peristalsis. Video imaging of colonic wall movements was used to make spatiotemporal maps and determine the velocity of peristalsis. Propagation of artificial fecal pellets in the guinea pig distal colon was studied in hexamethonium, atropine, ω-conotoxin (GVIA), ibodutant (MEN-15596), and TTX. Hexamethonium and ibodutant alone did not retard peristalsis. In contrast, ω-conotoxin abolished peristalsis in some preparations and reduced the velocity of propagation in all remaining specimens. Peristalsis could still occur in some animals in the presence of hexamethonium + atropine + ibodutant + ω-conotoxin. Peristalsis never occurred in the presence of TTX. The major finding of the current study is the unexpected observation that peristalsis can occur after blockade of the major excitatory neuroneuronal and neuromuscular transmitters. Also, the colon retained an intrinsic polarity in the presence of these antagonists and was only able to expel pellets in an aboral direction. The nature of the mechanism(s)/neurotransmitter(s) that generate(s) peristalsis and facilitate(s) natural fecal pellet propulsion, after blockade of major excitatory neurotransmitters, at the neuroneuronal and neuromuscular junction remains to be identified.