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 5 Hz and greater. Significant inhibition of CMCs required stimulation frequencies of 10 Hz and greater. Phentolamine (3.6 µM) 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.

Novel intrinsic neurogenic and myogenic mechanisms underlying the formation of faecal pellets along the large intestine of guinea-pigs.

Soft faecal material is transformed into discrete, pellet-shaped faeces at the colonic flexure. Here, analysis of water content in natural faecal material revealed a decline from cecum to rectum without significant changes at the flexure. Thus, pellet formation is not explained by changes in viscosity alone. We then used video imaging of colonic wall movements with electromyography in isolated preparations containing guinea-pig proximal colon, colonic flexure and distal colon. To investigate the pellet formation process, the colonic segments were infused with artificial contents (Krebs solution and 4-6% methylcellulose) to simulate physiological faecal content flow. Remarkably, pellet formation took place in vitro, without extrinsic neural inputs. Infusion evoked slowly propagating neurogenic contractions, the proximal colon migrating motor complexes (∼0.6 cpm), which initiated pellet formation at the flexure. Lesion of the flexure, but not the proximal colon, disrupted the formation of normal individual pellets. In addition, a distinct myogenic mechanism was identified, whereby slow phasic contractions (∼1.9 cpm) initiated at the flexure and propagated short distances retrogradely into the proximal colon and antegradely into the distal colon. There were no detectable changes in the density or distribution of pacemaker-type interstitial cells of Cajal across the flexure. The findings provide new insights into how solid faecal content is generated, suggesting the major mechanisms underlying faecal pellet formation involve the unique interaction at the colonic flexure between antegrade proximal colon migrating motor complexes, organized by enteric neurons, and retrograde myogenic slow phasic contractions. Additional, as yet unidentified extrinsic and/or humoral influences appear to contribute to processing of faecal content in vivo. KEY POINTS: In herbivores, including guinea-pigs, clearly defined faecal pellets are formed at a distinct location along the large intestine (colonic flexure). The mechanism underlying the formation of these faecal pellets at this region has remained unknown. We reveal a progressive and gradual reduction in water content of faecal content along the bowel. Hence, the distinct transition from amorphous to pellet shaped faecal content could not be explained by a dramatic increase in water reabsorption from a specific site. We discovered patterns of anterograde neurogenic and retrograde myogenic motor activity that facilitate the formation of faecal pellets. The formation of 'pellet-like' boluses at the colonic flexure involves interaction of an antegrade migrating motor complex in the proximal colon and retrograde myogenic slow phasic contractions that emerge from the colonic flexure. The findings uncover intrinsic mechanisms responsible for the formation of discrete faecal scybala in the large intestine of a vertebrate.

Motor patterns in the proximal and distal mouse colon which underlie formation and propulsion of feces.

BACKGROUND: In herbivores, the proximal and distal colonic regions feature distinct motor patterns underlying formation and propulsion of fecal pellets, respectively. Omnivores, such as mice and humans, lack a similar clear anatomical transition between colonic regions. We investigated whether distinct processes form and propel content along the large intestine of a mouse (an omnivore). METHODS: We recorded propulsive and non-propulsive neurogenic motor activity in mouse large intestine under six different stimulus conditions of varying viscosities. Gut wall movements were recorded by video and smooth muscle electrical behavior recorded with extracellular suction electrodes. KEY RESULTS: Three major neurally mediated motor patterns contributed to pellet formation and propulsion. (1) Pellet-shaped boluses are pinched off near the ceco-colonic junction and slowly propelled distally to a transition located at 40% length along the colon. (2) At this functional colonic flexure, propulsion speed is significantly increased by self-sustaining neural peristalsis. Speed transition at this location also occurs with artificial pellets and with spontaneously formed boluses in the empty colon. (3) Periodic colonic motor complexes (CMCs) were present in all conditions reaching a maximal frequency of about 0.4 cpm and extending across the proximal and distal colon with faster speed of propagation. CONCLUSIONS AND INFERENCES: The three motor patterns share a unique underlying fundamental property of the enteric circuits, which involve extended ensembles of enteric neurons firing at close to 2 Hz. The demonstration of distinct functional differences between proximal and distal colon in rabbit, guinea pig, and now mouse raises the possibility that this may be an organizational principle in other mammalian species, including humans.

Identification of neurogenic intestinal motility patterns in silver perch (Bidyanus bidyanus) that persist over wide temperature ranges.

BACKGROUND: Fish are increasingly being utilized as a model species for genetic manipulation studies related to gastrointestinal (GI) motility. Our aim was to identify whether patterns of GI motility in fish and the mechanisms underlying their generation are similar to those recorded from mammals (including humans). METHODS: The entire intestine was removed from euthanized adult Silver Perch (n = 11) and lesioned at the midway point to obtain two equal lengths. Proximal and distal segments were studied separately in organ baths with oxygenated Krebs solution, maintained at either 15°C (n = 5) or 25°C (n = 6). Motility was analyzed during rest, after oral infusion of Krebs solution, and after application of hexamethonium (100 µM) and tetrodotoxin (TTX) (0.6 µM). KEY RESULTS: Antegrade and retrograde propagating contractions (PC) were recorded in all preparations. In the proximal intestine, at 15 and 25°C, retrograde PCs occurred at 2.7 [1.7-4.5] and 3.1 [1.6-6.5] times the frequency of antegrade PCs, respectively. Colder temperatures did not inhibit PC frequency. Hexamethonium did not inhibit PC, and however, TTX abolished all contractile activity. CONCLUSIONS AND INFERENCES: Both neurogenic antegrade and retrograde propagating contractions occur throughout the intestine of Silver Perch. However, unlike the mammalian colon, these motor patterns do not require enteric nicotinic transmission and they are not inhibited by cold temperatures (15°C). Therefore, while the GI motility patterns in Silver Perch resemble those recorded from the colon of mammals, there may be differences in the mechanisms that underlying their generation.

Characterization of alternating neurogenic motor patterns in mouse colon.

BACKGROUND: Colonic motor complexes (CMCs) have been widely recorded in the large intestine of vertebrates. We have investigated whether in the smooth muscle, a single unified pattern of electrical activity, or different patterns of electrical activity give rise to the different neurogenic patterns of motility underlying CMCs in vitro. METHODS: To study differences of the CMCs between proximal and distal colon, we used a novel combination of techniques to simultaneously record muscle diameter and force at multiple sites along the whole mouse colon ex vivo. In addition, electrical activity of smooth muscle was recorded by suction electrodes. KEY RESULTS: Two distinct types of CMCs were distinguished; CMCs that propagated along the entire colon (complete CMC) and CMCs which were restricted to the proximal colon (incomplete CMC). The two types of CMC often occurred in the same preparations. Incomplete CMCs had longer bursts of smooth muscle action potentials than complete CMCs and propagated more slowly. Interestingly, both types of CMC were associated with similar frequency bursts of smooth muscle action potentials at ~2.4 Hz. In the most proximal colon, an additional firing frequency was detected close to ~7 Hz generating multiple peaks within each CMC. CONCLUSIONS & INFERENCES: We report distinct characteristics underlying complete and incomplete CMCs in isolated mouse colon. Recognizing these distinct patterns of motility will be important for future interpretation of analysis of murine colonic motility recordings. The identification of alternating patterns of motor activity in proximal colon, but not distal colon may reflect specific neural mechanisms for fecal pellet formation.

Neural motor complexes propagate continuously along the full length of mouse small intestine and colon.

Cyclical propagating waves of muscle contraction have been recorded in isolated small intestine or colon, referred to here as motor complexes (MCs). Small intestinal and colonic MCs are neurogenic, occur at similar frequencies, and propagate orally or aborally. Whether they can be coordinated between the different gut regions is unclear. Motor behavior of whole length mouse intestines, from duodenum to terminal rectum, was recorded by intraluminal multisensor catheter. Small intestinal MCs were recorded in 27/30 preparations, and colonic MCs were recorded in all preparations (n = 30) with similar frequencies (0.54 ± 0.03 and 0.58 ± 0.02 counts/min, respectively). MCs propagated across the ileo-colonic junction in 10/30 preparations, forming "full intestine" MCs. The cholinesterase inhibitor physostigmine increased the probability of a full intestine MC but had no significant effect on frequency, speed, or direction. Nitric oxide synthesis blockade by Nω-nitro-l-arginine, after physostigmine, increased MC frequency in small intestine only. Hyoscine-resistant MCs were recorded in the colon but not small intestine (n = 5). All MCs were abolished by hexamethonium (n = 18) or tetrodotoxin (n = 2). The enteric neural mechanism required for motor complexes is present along the full length of both the small and large intestine. In some cases, colonic MCs can be initiated in the distal colon and propagate through the ileo-colonic junction, all the way to duodenum. In conclusion, the ileo-colonic junction provides functional neural continuity for propagating motor activity that originates in the small or large intestine.NEW & NOTEWORTHY Intraluminal manometric recordings revealed motor complexes can propagate antegradely or retrogradely across the ileo-colonic junction, spanning the entire small and large intestines. The fundamental enteric neural mechanism(s) underlying cyclic motor complexes exists throughout the length of the small and large intestine.

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

KEY POINTS: 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. ABSTRACT: 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.

Functional changes in low- and high-threshold afferents in obstruction-induced bladder overactivity.

Neural mechanisms of lower urinary tract symptoms in obstruction-induced bladder overactivity remain unclear. We made the first single unit recordings from different types of spinal afferents to determine the effects of bladder outlet obstruction in guinea pigs. A model of gradual bladder outlet obstruction in male guinea pigs was used to produce overactive bladder. Conscious voiding was assessed in metabolic cages, and micturition was recorded in anesthetized guinea pigs in vivo. Single unit extracellular recordings were made ex vivo from spinal afferent nerves in flat sheet preparations of the bladder. Guinea pigs with partially obstructed bladders showed a significant increase in conscious voiding frequency compared with sham-operated guinea pigs. Also, nonvoiding contractions increased significantly in both frequency and amplitude. Although spontaneous firing of low-threshold bladder afferents was increased, their stretch-induced firing was reduced. The proportion of capsaicin-sensitive low-threshold afferents increased in obstructed bladders. Interestingly, spontaneous and stretch-induced firing were both significantly increased in high-threshold afferents after obstruction. In summary, sensory signaling increased in the obstructed bladder during the filling phase. This is largely mediated by low-threshold stretch-sensitive afferents that are activated by increased local nonvoiding contractions. Increased spontaneous firing by high-threshold afferents also contributes. Our findings revealed a complex effect of bladder outlet obstruction on different types of bladder afferents that needs consideration for potential therapeutic targeting of lower urinary tract symptoms in obstruction-induced bladder overactivity.

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.