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.

Duodenal and proximal jejunal motility inhibition associated with bisacodyl-induced colonic high-amplitude propagating contractions.

Bisacodyl is a stimulant laxative often used in manometric studies of pediatric constipation to determine if it can initiate propulsive high-amplitude propagating contractions (HAPCs). Whereas the effects of bisacodyl infusion on colonic motility are well described, the effects of the drug on other regions of the gut after colonic infusion are not known. The aim of the present study was to characterize the effects of bisacodyl on both colonic and small bowel motility. Twenty-seven children (9.3 ± 1.2 yr) undergoing simultaneous high-resolution antroduodenal and colonic manometry were included. Small bowel and colonic motor patterns were assessed before and after colonic infusion of bisacodyl. Patients were divided into two groups: responders and nonresponders based on the presence of high-amplitude propagating contractions (HAPCs) after bisacodyl infusion. Nineteen patients were responders. A total of 188 postbisacodyl HAPCs was identified with a mean count of 10.4 ± 5.5 (range, 3-22), at a frequency of 0.6 ± 0.2/min and mean amplitude of 119.8 ± 23.6 mmHg. No motor patterns were induced in the small bowel. However, in the 19 responders the onset of HAPCs was associated with a significant decrease in small bowel contractile activity. In the nonresponders, there was no detectable change in small bowel motility after bisacodyl infusion. Bisacodyl-induced HAPCs are associated with a significant reduction in small bowel motility probably mediated by extrinsic sympathetic reflex pathways. This inhibition is potentially related to rectal distension, caused by the HAPC anal propulsion of colonic content.NEW & NOTEWORTHY The present study has shown, for the first time, that the presence of high-amplitude propagating contractions induced by bisacodyl is associated with a significant reduction in small bowel motility. These findings support of possible existence of a reflex pathway that causes inhibition of small bowel motility in response to rectal distension.

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.

Synaptic activation of putative sensory neurons by hexamethonium-sensitive nerve pathways in mouse colon.

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5-10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 µM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways. NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways. Putative sensory Dogiel type II calcitonin gene-related peptide-immunoreactive myenteric neurons may receive widespread fast synaptic inputs in mouse colon.

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.

Identification of different functional types of spinal afferent neurons innervating the mouse large intestine using a novel CGRPα transgenic reporter mouse.

Spinal afferent neurons detect noxious and physiological stimuli in visceral organs. Five functional classes of afferent terminals have been extensively characterized in the colorectum, primarily from axonal recordings. Little is known about the corresponding somata of these classes of afferents, including their morphology, neurochemistry, and electrophysiology. To address this, we made intracellular recordings from somata in L6/S1 dorsal root ganglia and applied intraluminal colonic distensions. A transgenic calcitonin gene-related peptide-α (CGRPα)-mCherry reporter mouse, which enabled rapid identification of soma neurochemistry and morphology following electrophysiological recordings, was developed. Three distinct classes of low-threshold distension-sensitive colorectal afferent neurons were characterized; an additional group was distension-insensitive. Two of three low-threshold classes expressed CGRPα. One class expressing CGRPα discharged phasically, with inflections on the rising phase of their action potentials, at low frequencies, to both physiological (<30 mmHg) and noxious (>30 mmHg) distensions. The second class expressed CGRPα and discharged tonically, with smooth, briefer action potentials and significantly greater distension sensitivity than phasically firing neurons. A third class that lacked CGRPα generated the highest-frequency firing to distension and had smaller somata. Thus, CGRPα expression in colorectal afferents was associated with lower distension sensitivity and firing rates and larger somata, while colorectal afferents that generated the highest firing frequencies to distension had the smallest somata and lacked CGRPα. These data fill significant gaps in our understanding of the different classes of colorectal afferent somata that give rise to distinct functional classes of colorectal afferents. In healthy mice, the majority of sensory neurons that respond to colorectal distension are low-threshold, wide-dynamic-range afferents, encoding both physiological and noxious ranges.

Rectal prolapse in Winnie mice with spontaneous chronic colitis: changes in intrinsic and extrinsic innervation of the rectum.

Rectal prolapse is associated with diminished anal sensitivity and rectal motor activity. Both sensory and motor functions are controlled by the extrinsic and intrinsic (enteric nervous system) innervation of the gastrointestinal tract. Studies of changes in intestinal innervation in humans and in animal models with rectal prolapse are extremely scarce. The Winnie mouse model of spontaneous chronic colitis closely represents human inflammatory bowel disease and is prone to develop rectal prolapse. We have investigated changes in the myenteric and inhibitory motor neurons and evaluated changes in the density of sensory afferent, sympathetic, and parasympathetic fibers in the rectal colon of Winnie mice with and without rectal prolapse. Our results demonstrate that rectal prolapse in Winnie mice with chronic colitis is correlated with enhanced levels of inflammation, gross morphological damage, and muscular hypertrophy of the rectum. Animals with prolapse have more severe damage to the rectal innervation compared with Winnie mice without prolapse. This includes more severe neuronal loss in the myenteric plexus, involving a decrease in nNOS-immunoreactive neurons (not observed in Winnie mice without prolapse) and a more pronounced loss of VAChT-immunoreactive fibers. Both Winnie mice with and without prolapse have comparable levels of noradrenergic and sensory fiber loss in the rectum. This is the first study providing evidence that the damage and death of enteric neurons, including nitrergic neurons in myenteric ganglia and the loss of cholinergic nerve fibers, are important factors in structural changes in the rectum of mice with rectal prolapse.

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

The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca(2+) imaging, immunocytochemistry and 3D modelling. Ca(2+) enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca(2+) channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.

Electrophysiological and morphological features of myenteric neurons of human colon revealed by intracellular recording and dye fills.

BACKGROUND: Ex vivo intracellular recordings and dye fills, combined with immunohistochemistry, are a powerful way to analyze the enteric nervous system of laboratory animals. METHODS: Myenteric neurons were recorded in isolated specimens of human colon. A key determinant of successful recording was near-complete removal of circular muscle from the surface of ganglia. KEY RESULTS: Treatment with a collagenase/neutral protease mix before dissection significantly improved recording success and reduced damage to the plexus. Carboxyfluorescein in microelectrodes allowed recorded neurons to be routinely labeled, analyzed, and subjected to multi-layer immunohistochemistry. Carboxyfluorescein revealed morphological details that were not detected by immunohistochemical methods. Of 54 dye-filled myenteric neurons (n = 22), 45 were uni-axonal and eight were multi-axonal. There was a significant bias toward recordings from large neural somata. The close association between morphology and electrophysiology (long after-hyperpolarizations and fast EPSPs) seen in mice and guinea pigs did not hold for human myenteric neuron recordings. No slow EPSPs were recorded; however, disruption to the myenteric plexus during dissection may have led the proportion of cells receiving synaptic potentials to be underestimated. Neurons immunoreactive for nitric oxide synthase were more excitable than non-immunoreactive neurons. Distinctive grooves were observed on the serosal and/or mucosal faces of myenteric neurons in 3D reconstructions. These had varicose axons running through them and may represent a preferential site of synaptic inputs. CONCLUSIONS: Human enteric neurons share many features with laboratory animals, but the combinations of features in individual cells appear more variable.

Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding.

BACKGROUND AND AIMS: Gut functions including motility, secretion, and blood flow are largely controlled by the enteric nervous system. Characterizing the different classes of enteric neurons in the human gut is an important step to understand how its circuitry is organized and how it is affected by disease. METHODS: Using multiplexed immunohistochemistry, 12 discriminating antisera were applied to distinguish different classes of myenteric neurons in the human colon (2596 neurons, 12 patients) according to their chemical coding. All antisera were applied to every neuron, in multiple layers, separated by elutions. RESULTS: A total of 164 combinations of immunohistochemical markers were present among the 2596 neurons, which could be divided into 20 classes, with statistical validation. Putative functions were ascribed for 4 classes of putative excitatory motor neurons (EMN1-4), 4 inhibitory motor neurons (IMN1-4), 3 ascending interneurons (AIN1-3), 6 descending interneurons (DIN1-6), 2 classes of multiaxonal sensory neurons (SN1-2), and a small, miscellaneous group (1.8% of total). Soma-dendritic morphology was analyzed, revealing 5 common shapes distributed differentially between the 20 classes. Distinctive baskets of axonal varicosities surrounded 45% of myenteric nerve cell bodies and were associated with close appositions, suggesting possible connectivity. Baskets of cholinergic terminals and several other types of baskets selectively targeted ascending interneurons and excitatory motor neurons but were significantly sparser around inhibitory motor neurons. CONCLUSIONS: Using a simple immunohistochemical method, human myenteric neurons were shown to comprise multiple classes based on chemical coding and morphology and dense clusters of axonal varicosities were selectively associated with some classes.

Characterization of viscerofugal neurons in human colon by retrograde tracing and multi-layer immunohistochemistry.

Background and Aims: Viscerofugal neurons (VFNs) have cell bodies in the myenteric plexus and axons that project to sympathetic prevertebral ganglia. In animals they activate sympathetic motility reflexes and may modulate glucose metabolism and feeding. We used rapid retrograde tracing from colonic nerves to identify VFNs in human colon for the first time, using ex vivo preparations with multi-layer immunohistochemistry. Methods: Colonic nerves were identified in isolated preparations of human colon and set up for axonal tracing with biotinamide. After fixation, labeled VFN cell bodies were subjected to multiplexed immunohistochemistry for 12 established nerve cell body markers. Results: Biotinamide tracing filled 903 viscerofugal nerve cell bodies (n = 23), most of which (85%) had axons projecting orally before entering colonic nerves. Morphologically, 97% of VFNs were uni-axonal. Of 215 VFNs studied in detail, 89% expressed ChAT, 13% NOS, 13% calbindin, 9% enkephalin, 7% substance P and 0 of 123 VFNs expressed CART. Few VFNs contained calretinin, VIP, 5HT, CGRP, or NPY. VFNs were often surrounded by dense baskets of axonal varicosities, probably reflecting patterns of connectivity; VAChT+ (cholinergic), SP+ and ENK+ varicosities were most abundant around them. Human VFNs were diverse; showing 27 combinations of immunohistochemical markers, 4 morphological types and a wide range of cell body sizes. However, 69% showed chemical coding, axonal projections, soma-dendritic morphology and connectivity similar to enteric excitatory motor neurons. Conclusion: Viscerofugal neurons are present in human colon and show very diverse combinations of features. High proportions express ChAT, consistent with cholinergic synaptic outputs onto postganglionic sympathetic neurons in prevertebral ganglia.

Loss of responsiveness of circular smooth muscle cells from the guinea pig ileum is associated with changes in gap junction coupling.

Gap junction coupling and neuromuscular transmission to smooth muscle were studied in the first 4 h after preparations were set up in vitro. Intracellular recordings were made from smooth muscle cells of guinea pig ileum. Fast inhibitory junction potentials (IJPs) were small (1.3 ± 1.0 mV) in the first 30 min but increased significantly over the first 120 min to 15.8 ± 0.9 mV (n = 12, P < 0.001). Comparable increases in slow IJPs and excitatory junction potentials were also observed. During the same period, resting membrane potential depolarized from -58.8 ± 1.4 to -47.2 ± 0.4 mV (n = 12, P < 0.001). Input resistance, estimated by intracellular current injection, decreased in parallel (P < 0.05), and dye coupling, measured by intracellular injection of carboxyfluorescein, increased (P < 0.001). Input resistance was higher and dye coupling was less in longitudinal than circular smooth muscle cells. Gap junction blockers [carbenoxolone (100 µM), 18ß-glycyrrhetinic acid (10 µM), and 2-aminoethoxydiphenyl borate (50 µM)] hyperpolarized coupled circular smooth muscle cells, reduced the amplitude of fast and slow IJPs and excitatory junction potentials, increased input resistance, and reduced dye coupling. Local application of ATP (10 mM) mimicked IJPs and showed comparable increases in amplitude over the first 120 min; carbenoxolone and 2-aminoethoxydiphenyl borate significantly reduced ATP-evoked hyperpolarizations in coupled cells. In contrast, synaptic transmission between myenteric neurons was not suppressed during the first 30 min. Gap junction coupling between circular smooth muscle cells in isolated preparations was initially disrupted but recovered over the next 120 min to a steady level. This was associated with potent effects on neuromuscular transmission and responses to exogenous ATP.

Sympathetic Pathways Target Cholinergic Neurons in the Human Colonic Myenteric Plexus.

Background: The sympathetic nervous system inhibits human colonic motility largely by effects on enteric neurons. Noradrenergic axons, which branch extensively in the myenteric plexus, are integral to this modulatory role, but whether they contact specific types of enteric neurons is unknown. The purpose of this study was to determine the association of noradrenergic varicosities with types of enteric neurons. Methods: Human colonic tissue from seven patients was fixed and dissected prior to multi-layer immunohistochemistry for human RNA binding proteins C and D (HuC/D) (pan-neuronal cell body labelling), tyrosine hydroxylase (TH, catecholaminergic labelling), Enkephalin (ENK), choline acetyltransferase (ChAT, cholinergic labelling) and/or nitric oxide synthase (NOS, nitrergic labelling) and imaged using confocal microscopy. TH-immunoreactive varicose nerve endings and myenteric cell bodies were reconstructed as three dimensional digital images. Data was exported to a purpose-built software package which quantified the density of varicosities close to the surface of each myenteric cell body. Results: TH-immunoreactive varicosities had a greater mean density within 1 µm of the surface of ChAT +/NOS- nerve cell bodies compared with ChAT-/NOS + cell bodies. Similarly, ENK-immunoreactive varicosities also had a greater mean density close to ChAT +/NOS- cell bodies compared with ChAT-/NOS + cells. Conclusion: A method for quantifying close associations between varicosities and nerve cell bodies was developed. Sympathetic axons in the myenteric plexus preferentially target cholinergic excitatory cells compared to nitrergic neurons (which are largely inhibitory). This connectivity is likely to be involved in inhibitory modulation of human colonic motility by the sympathetic nervous system.

Characterisation of parasympathetic ascending nerves in human colon.

Background: In the human large bowel, sacral parasympathetic nerves arise from S2 to S4, project to the pelvic plexus ("hypogastric plexus") and have post-ganglionic axons entering the large bowel near the rectosigmoid junction. They then run long distances orally or aborally within the bowel wall forming "ascending nerves" or "shunt fascicles" running in the plane of the myenteric plexus. They form bundles of nerve fibres that can be distinguished from the myenteric plexus by their straight orientation, tendency not to merge with myenteric ganglia and greater width. Aim: To identify reliable marker(s) to distinguish these bundles of ascending nerves from other extrinsic and intrinsic nerves in human colon. Methods: Human colonic segments were obtained with informed consent, from adult patients undergoing elective surgery (n = 21). Multi-layer immunohistochemical labelling with neurofilament-H (NF200), myelin basic protein (MBP), von Willebrand factor (vWF), and glucose transporter 1 (GLUT1), and rapid anterograde tracing with biotinamide, were used to compare ascending nerves and lumbar colonic nerves. Results: The rectosigmoid and rectal specimens had 6-11 ascending nerves spaced around their circumference. Distal colon specimens typically had 1-3 ascending nerves, with one located near the mesenteric taenia coli. No ascending nerves were observed in ascending colon specimens. GLUT1 antisera labelled both sympathetic lumbar colonic nerves and ascending nerves in the gut wall. Lumbar colonic nerves joined the myenteric plexus and quickly lost GLUT1 labelling, whereas GLUT1 staining labelled parasympathetic ascending nerves over many centimetres. Conclusion: Ascending nerves can be distinguished in the colorectum of humans using GLUT1 labelling combined with NF200.

The role of enteric inhibitory neurons in intestinal motility.

The enteric nervous system controls much of the mixing and propulsion of nutrients along the digestive tract. Enteric neural circuits involve intrinsic sensory neurons, interneurons and motor neurons. While the role of the excitatory motor neurons is well established, the role of the enteric inhibitory motor neurons (IMNs) is less clear. The discovery of inhibitory transmission in the intestine in the 1960's in the laboratory of Geoff Burnstock triggered the search for the unknown neurotransmitter. It has since emerged that most neurons including the IMNs contain and may utilise more than one transmitter substances; for IMNs these include ATP, the neuropeptide VIP/PACAP and nitric oxide. This review distinguishes the enteric neural pathways underlying the 'standing reflexes' from the pathways operating physiologically during propulsive and non-propulsive movements. Morphological evidence in small laboratory animals indicates that the IMNs are located in the myenteric plexus and project aborally to the circular muscle, where they act by relaxing the muscle. There is ongoing 'tonic' activity of these IMNs to keep the intestinal muscle relaxed. Accommodatory responses to content further activate enteric pathways that involve the IMNs as the final neural element. IMNs are activated by mechanical and chemical stimulation induced by luminal contents, which activate intrinsic sensory enteric neurons and the polarised interneuronal ascending excitatory and descending inhibitory reflex pathways. The latter relaxes the muscle ahead of the advancing bolus, thus facilitating propulsion.

Characterization of putative interneurons in the myenteric plexus of human colon.

BACKGROUND: The enteric nervous system contains multiple classes of neurons, distinguishable by morphology, immunohistochemical markers, and projections; however, specific combinations differ between species. Here, types of enteric neurons in human colon were characterized immunohistochemically, using retrograde tracing combined with multiple labeling immunohistochemistry, focussing on non-motor neurons. METHODS: The fluorescent carbocyanine tracer, DiI, was applied to the myenteric plexus in ex vivo preparations, filling neurons projecting within the plexus. Limits of projection lengths of motor neurons were established, allowing them to be excluded from the analysis. Long ascending and descending interneurons were then distinguished by labeling for discriminating immunohistochemical markers: calbindin, calretinin, enkephalin, 5-hydroxytryptamine, nitric oxide synthase, and substance P. These results were combined with a previous published study in which nitric oxide synthase and choline acetyltransferase immunoreactivities were established. KEY RESULTS: Long ascending neurons (with projections longer than 8 mm, which excludes more than 95% motor neurons) formed four types, in descending order of abundance, defined by immunoreactivity for: (a) ChAT+/ENK+, (b) ChAT+/ENK+/SP+, (c) ChAT+/Calb+, and (d) ChAT+/ENK+/Calb+. Long descending neurons, up to 70 mm long also formed at least four types, distinguished by immunoreactivity for (a) NOS + cells (without ChAT), (b) ChAT+/NOS+, (c) ChAT+/Calret+, and (d) ChAT+/5HT + cells (with or without NOS). CONCLUSIONS AND INFERENCES: Long interneurons, which do not innervate muscularis externa, are likely to coordinate neural activity over distances of many centimeters along the colon. Characterizing their neurochemical coding provides a basis for understanding their roles, investigating their connectivity, and building a comprehensive account of human colonic enteric neurons.

Identification of medium/high-threshold extrinsic mechanosensitive afferent nerves to the gastrointestinal tract.

BACKGROUND & AIMS: Large distentions reliably evoke sensation from the noninflamed, nonischemic bowel, but the specialized afferent axonal structures responsible have not been morphologically identified. We investigated whether their transduction sites are located on major blood vessels close to and within the gut wall. METHODS: In vitro extracellular recordings were made from mesenteric nerve trunks in guinea pig ileum, combined with rapid axonal dye filling and immunohistochemical analysis of nerve trunks. RESULTS: Recordings revealed sensory fibers with focal mechanosensitive sites in the mesenteries that could be activated by von Frey hairs and by stretch. Dye filling revealed varicose branching sensory axons on mesenteric blood vessels but no other anatomically specialized structures in mesenteric membranes or the serosa. Large-amplitude stretch and von Frey hairs also activated sensory endings within the gut wall itself but only if the submucosa was present; mechanotransduction sites in the serosa or outer muscle layers were sparse. Mechanosensitive sites in submucosa were exclusively associated with submucosal blood vessels. Submucosal endings had significantly higher thresholds to stretch than specialized low-threshold mechanoreceptors characterized previously in the rectum (P < .05) and were therefore classified as medium/high-threshold mechanoreceptors. Capsaicin (0.3-1 micromol/L) activated most mechanosensitive mesenteric (68%) and submucosal (85%) afferent endings. Similar intramural mechanosensitive afferent endings on blood vessels also exist in the colon and bladder. CONCLUSIONS: Varicose branching axons of sensory neurons on intramural blood vessels, previously shown to mediate sensory vasodilation, are transduction sites for medium/high-threshold, stretch-sensitive mechanoreceptors, encoding large distentions in hollow viscera.