Nick J. Spencer

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

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

Mechanisms underlying distension-evoked peristalsis in guinea pig distal colon: is there a role for enterochromaffin cells?

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.

Identification of the Visceral Pain Pathway Activated by Noxious Colorectal Distension in Mice

In patients with irritable bowel syndrome, visceral pain is evoked more readily following distension of the colorectum. However, the identity of extrinsic afferent nerve pathway that detects and transmits visceral pain from the colorectum to the spinal cord is unclear. In this study, we identified which extrinsic nerve pathway(s) underlies nociception from the colorectum to the spinal cord of rodents. Electromyogram recordings were made from the transverse oblique abdominal muscles in anesthetized wild type (C57BL/6) mice and acute noxious intraluminal distension stimuli (100–120 mmHg) were applied to the terminal 15 mm of colorectum to activate visceromotor responses (VMRs). Lesioning the lumbar colonic nerves in vivo had no detectable effect on the VMRs evoked by colorectal distension. Also, lesions applied to the right or left hypogastric nerves failed to reduce VMRs. However, lesions applied to both left and right branches of the rectal nerves abolished VMRs, regardless of whether the lumbar colonic or hypogastric nerves were severed. Electrical stimulation applied to either the lumbar colonic or hypogastric nerves in vivo, failed to elicit a VMR. In contrast, electrical stimulation (2–5 Hz, 0.4 ms, 60 V) applied to the rectum reliably elicited VMRs, which were abolished by selective lesioning of the rectal nerves. DiI retrograde labeling from the colorectum (injection sites 9–15 mm from the anus, measured in unstretched preparations) labeled sensory neurons primarily in dorsal root ganglia (DRG) of the lumbosacral region of the spinal cord (L6-S1). In contrast, injection of DiI into the mid to proximal colon (injection sites 30–75 mm from the anus, measured in unstretched preparations) labeled sensory neurons in DRG primarily of the lower thoracic level (T6-L2) of the spinal cord. The visceral pain pathway activated by acute noxious distension of the terminal 15 mm of mouse colorectum is transmitted predominantly, if not solely, through rectal/pelvic afferent nerve fibers to the spinal cord. The sensory neurons of this spinal afferent pathway lie primarily in the lumbosacral region of the spinal cord, between L6 and S1.

Mechanotransduction and chemosensitivity of two major classes of bladder afferents with endings in the vicinity to the urothelium

The guinea pig bladder is innervated by at least five distinct major classes of extrinsic sensory neurons. In this study, we have examined the mechanisms of mechanotransduction and chemosensitivity of two classes of bladder afferents that have their endings in the vicinity of the urothelium: stretch‐sensitive muscle‐mucosal mechanoreceptors and stretch‐insensitive, mucosal high‐responding afferents. The non‐selective P2 purinoreceptor antagonist pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid did not affect stretch‐ or stroking‐induced firing of these afferents but significantly reduced the excitatory action of α,β‐methylene ATP. Blocking synaptic transmission in Ca2+‐free solution did not affect stretch‐evoked firing but slightly reduced stretch‐induced tension responses. Stroking‐induced firing of both classes of afferents was also not affected in Ca2+‐free solution. Of blockers of mechano‐gated channels, benzamil (100 μm), but not amiloride (100 μm), Gd3+ (100 μm) or SKF 96365 (50 μm), inhibited stretch‐ and stroking‐induced firing. Serotonin (100 μm) applied directly onto receptive fields predominantly activated muscle‐mucosal afferents. Muscarine (100 μm) and substance P (100 μm) in 24% and 36% cases activated only mucosal high‐responding units. Bradykinin (10 μm), but not prostaglandin E2 (10 μm), excites predominantly mucosal units. High (80 mm) K+ solution activated both afferent classes, but responses of mucosal units were 4 times greater. In contrast to muscle‐mucosal units, most mucosal high‐responding units were activated by hot Krebs solution (45–46°C), low pH (pH 4) and capsaicin (3 μm). TRPV1 antagonist, capsazepine (10 μm) was without effect on mechanotransduction by mucosal high‐responding afferents. The results show that mechanotransduction of these two types of afferents are not dependant upon Ca2+‐dependent exocytotic release of mediators, or ATP, and it is likely that benzamil‐sensitive stretch‐activated ion channels on their endings are involved in direct mechanotransduction. The chemosensitivity to agonists and noxious stimuli differs significantly between these two major classes of bladder afferents that reflects their different physiological and pathophysiological roles in the bladder.

An experimental method to identify neurogenic and myogenic active mechanical states of intestinal motility

Excitatory and inhibitory enteric neural input to intestinal muscle acting on ongoing myogenic activity determines the rich repertoire of motor patterns involved in digestive function. The enteric neural activity cannot yet be established during movement of intact intestine in vivo or in vitro. We propose the hypothesis that is possible to deduce indirectly, but reliably, the state of activation of the enteric neural input to the muscle from measurements of the mechanical state of the intestinal muscle. The fundamental biomechanical model on which our hypothesis is based is the “three-element model” proposed by Hill. Our strategy is based on simultaneous video recording of changes in diameters and intraluminal pressure with a fiber-optic manometry in isolated segments of rabbit colon. We created a composite spatiotemporal map (DPMap) from diameter (DMap) and pressure changes (PMaps). In this composite map rhythmic myogenic motor patterns can readily be distinguished from the distension induced neural peristaltic contractions. Plotting the diameter changes against corresponding pressure changes at each location of the segment, generates “orbits” that represent the state of the muscle according to its ability to contract or relax actively or undergoing passive changes. With a software developed in MatLab, we identified twelve possible discrete mechanical states and plotted them showing where the intestine actively contracted and relaxed isometrically, auxotonically or isotonically, as well as where passive changes occurred or was quiescent. Clustering all discrete active contractions and relaxations states generated for the first time a spatio-temporal map of where enteric excitatory and inhibitory neural input to the muscle occurs during physiological movements. Recording internal diameter by an impedance probe proved equivalent to measuring external diameter, making possible to further develop similar strategy in vivo and humans.

Structure–function relationship of sensory endings in the gut and bladder

Visceral afferents play a key role in neural circuits underlying the physiological function of visceral organs. They are responsible for the detection and transmission of a variety of visceral sensations (e.g. satiety, urge, discomfort and pain) from the viscera to the central nervous system. A comprehensive account of the different functional types of visceral sensory neurons would be invaluable in understanding how sensory dysfunction occurs and how it might be diagnosed and treated. Our aim was to explore the morphology of different nerve endings of visceral afferents within the gastrointestinal tract and urinary bladder and how the morphology of these nerve endings may relate to their functional properties. Morphological studies of mechanosensitive endings of visceral afferents to the gut and bladder correlated with physiological recordings have added a new dimension to our ability to distinguish different functional classes of visceral afferents.

Neurogenic and myogenic motor patterns of rabbit proximal, mid, and distal colon

The rabbit colon consists of four distinct regions. The motility of each region is controlled by myogenic and neurogenic mechanisms. Associating these mechanisms with specific motor patterns throughout all regions of the colon has not previously been achieved. Three sections of the colon (the proximal, mid, and distal colon) were removed from euthanized rabbits. The proximal colon consists of a triply teniated region and a single tenia region. Spatio-temporal maps were constructed from video recordings of colonic wall diameter, with associated intraluminal pressure recorded from the aboral end. Hexamethonium (100 μM) and tetrodotoxin (TTX; 0.6 μM) were used to inhibit neural activity. Four distinct patterns of motility were detected: 1 myogenic and 3 neurogenic. The myogenic activity consisted of circular muscle (CM) contractions (ripples) that occurred throughout the colon and propagated in both antegrade (anal) and retrograde (oral) directions. The neural activity of the proximal colon consisted of slowly (0.1 mm/s) propagating colonic migrating motor complexes, which were abolished by hexamethonium. These complexes were observed in the region of the proximal colon with a single band of tenia. In the distal colon, tetrodotoxin-sensitive, thus neurally mediated, but hexamethonium-resistant, peristaltic (anal) and antiperistaltic (oral) contractions were identified. The distinct patterns of neurogenic and myogenic motor activity recorded from isolated rabbit colon are specific to each anatomically distinct region. The regional specificity motor pattern is likely to facilitate orderly transit of colonic content from semi-liquid to solid composition of feces.

Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells

•  Enterochromaffin (EC) cells are enteroendocrine cells that synthesise ∼95% of the bodys total serotonin (5‐HT). •  Although 5‐HT release from EC cells plays a number of important physiological roles, primary EC cells have not been studied at the single cell level. •  This study provides the first functional characterisation of single primary guinea‐pig and human EC cells. •  EC cells release 5‐HT from large dense core vesicles in a calcium‐dependent manner with kinetics surprisingly resembling release from synaptic vesicles. •  3D modelling indicates that the quantity of 5‐HT released per vesicle fusion event is physiologically relevant to GI tract function in terms of the concentrations needed to activate local 5‐HT receptors. •  These findings represent significant advances in our understanding of EC cell function and will be of broad interest to researchers in endocrine cell biology, gastroenterology, neuroscience, exocytosis and glucose control.

Identification of functional intramuscular rectal mechanoreceptors in aganglionic rectal smooth muscle from piebald lethal mice

The mechanosensitive endings of low-threshold, slowly adapting pelvic afferents that innervate the rectum have been previously identified as rectal intraganglionic laminar endings (rIGLEs) that lie within myenteric ganglia. We tested whether the aganglionic rectum of piebald-lethal (s(l)/s(l)) mice lacks rIGLEs and whether this could explain impaired distension-evoked reflexes from this region. Extracellular recordings were made from fine rectal nerves in C57BL/6 wild-type and s(l)/s(l) mice, combined with anterograde labeling. In C57BL/6 mice, graded circumferential stretch applied to the rectum activated graded increases in firing of slowly adapting rectal mechanoreceptors. In s(l)/s(l) mice, graded stretch of the aganglionic rectum activated similar graded increases in rectal afferent firing. Stretch-sensitive afferents responded at low mechanical thresholds and fired more intensely at noxious levels of stretch. They could also be activated by probing their receptive fields with von Frey hairs and by muscle contraction. Anterograde labeling from recorded rectal nerves identified the mechanoreceptors of muscular afferents in the aganglionic rectal smooth muscle. A population of afferents were also recorded in both C57BL/6 and s(l)/s(l) mice that were activated by von Frey hair probing, but not stretch. In summary, the aganglionic rectum is innervated by a population of stretch-sensitive rectal afferent mechanoreceptor which develops and functions in the absence of any enteric ganglia. These results suggest that in patients with Hirschsprungs disease the inability to activate extrinsic distension reflexes from the aganglionic rectum is unlikely to be due to the absence of stretch-sensitive extrinsic mechanoreceptors.

Loss of visceral pain following colorectal distension in an endothelin-3 deficient mouse model of Hirschsprung's disease

Non‐technical summary  The endothelin‐3 (ET‐3) gene is essential for the development of the enteric nervous system in the gastrointestinal tract of mammals, including humans and mice. Loss of the ET‐3 gene leads to the formation of an aganglionic colorectum and impaired bowel function. Endogenous endothelin peptides and their receptors also play a major role in nociception in a variety of organs and species, including humans. However, whether nociception is altered in the aganglionic region of the colorectum is unknown. We show that in ET‐3 deficient mice, there is a loss of nociception from the aganglionic rectum, but not other visceral organs. This loss of nociception is due to a reduction in spinal afferent innervation and a selective deficiency in specific classes of rectal afferent nerve fibres, which are necessary for detection of noxious stimuli from this region.