A Gut-Intrinsic Melanocortin Signaling Complex Augments L-Cell Secretion in Humans.

OBJECTIVE: Hypothalamic melanocortin 4 receptors (MC4R) are a key regulator of energy homeostasis. Brain-penetrant MC4R agonists have failed, as concentrations required to suppress food intake also increase blood pressure. However, peripherally located MC4R may also mediate metabolic benefits of MC4R activation. Mc4r transcript is enriched in mouse enteroendocrine L cells and peripheral administration of the endogenous MC4R agonist, α-melanocyte stimulating hormone (α-MSH), triggers the release of the anorectic hormones Glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in mice. This study aimed to determine whether pathways linking MC4R and L-cell secretion exist in humans. DESIGN: GLP-1 and PYY levels were assessed in body mass index-matched individuals with or without loss-of-function MC4R mutations following an oral glucose tolerance test. Immunohistochemistry was performed on human intestinal sections to characterize the mucosal MC4R system. Static incubations with MC4R agonists were carried out on human intestinal epithelia, GLP-1 and PYY contents of secretion supernatants were assayed. RESULTS: Fasting PYY levels and oral glucose-induced GLP-1 secretion were reduced in humans carrying a total loss-of-function MC4R mutation. MC4R was localized to L cells and regulates GLP-1 and PYY secretion from ex vivo human intestine. α-MSH immunoreactivity in the human intestinal epithelia was predominantly localized to L cells. Glucose-sensitive mucosal pro-opiomelanocortin cells provide a local source of α-MSH that is essential for glucose-induced GLP-1 secretion in small intestine. CONCLUSION: Our findings describe a previously unidentified signaling nexus in the human gastrointestinal tract involving α-MSH release and MC4R activation on L cells in an autocrine and paracrine fashion. Outcomes from this study have direct implications for targeting mucosal MC4R to treat human metabolic disorders.

Identification of a Rhythmic Firing Pattern in the Enteric Nervous System That Generates Rhythmic Electrical Activity in Smooth Muscle.

The enteric nervous system (ENS) contains millions of neurons essential for organization of motor behavior of the intestine. It is well established that the large intestine requires ENS activity to drive propulsive motor behaviors. However, the firing pattern of the ENS underlying propagating neurogenic contractions of the large intestine remains unknown. To identify this, we used high-resolution neuronal imaging with electrophysiology from neighboring smooth muscle. Myoelectric activity underlying propagating neurogenic contractions along murine large intestine [also referred to as colonic migrating motor complexes, (CMMCs)] consisted of prolonged bursts of rhythmic depolarizations at a frequency of ∼2 Hz. Temporal coordination of this activity in the smooth muscle over large spatial fields (∼7 mm, longitudinally) was dependent on the ENS. During quiescent periods between neurogenic contractions, recordings from large populations of enteric neurons, in mice of either sex, revealed ongoing activity. The onset of neurogenic contractions was characterized by the emergence of temporally synchronized activity across large populations of excitatory and inhibitory neurons. This neuronal firing pattern was rhythmic and temporally synchronized across large numbers of ganglia at ∼2 Hz. ENS activation preceded smooth muscle depolarization, indicating rhythmic depolarizations in smooth muscle were controlled by firing of enteric neurons. The cyclical emergence of temporally coordinated firing of large populations of enteric neurons represents a unique neural motor pattern outside the CNS. This is the first direct observation of rhythmic firing in the ENS underlying rhythmic electrical depolarizations in smooth muscle. The pattern of neuronal activity we identified underlies the generation of CMMCs.SIGNIFICANCE STATEMENT How the enteric nervous system (ENS) generates neurogenic contractions of smooth muscle in the gastrointestinal (GI) tract has been a long-standing mystery in vertebrates. It is well known that myogenic pacemaker cells exist in the GI tract [called interstitial cells of Cajal (ICCs)] that generate rhythmic myogenic contractions. However, the mechanisms underlying the generation of rhythmic neurogenic contractions of smooth muscle in the GI tract remains unknown. We developed a high-resolution neuronal imaging method with electrophysiology to address this issue. This technique revealed a novel pattern of rhythmic coordinated neuronal firing in the ENS that has never been identified. Rhythmic neuronal firing in the ENS was found to generate rhythmic neurogenic depolarizations in smooth muscle that underlie contraction of the GI tract.

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.

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.

Augmented capacity for peripheral serotonin release in human obesity.

BACKGROUND/OBJECTIVES: Evidence from animal studies highlights an important role for serotonin (5-HT), derived from gut enterochromaffin (EC) cells, in regulating hepatic glucose production, lipolysis and thermogenesis, and promoting obesity and dysglycemia. Evidence in humans is limited, although elevated plasma 5-HT concentrations are linked to obesity. SUBJECTS/METHODS: We assessed (i) plasma 5-HT concentrations before and during intraduodenal glucose infusion (4 kcal/min for 30 min) in non-diabetic obese (BMI 44 ± 4 kg/m2, N = 14) and control (BMI 24 ± 1 kg/m2, N = 10) subjects, (ii) functional activation of duodenal EC cells (immunodetection of phospho-extracellular related-kinase, pERK) in response to glucose, and in separate subjects, (iii) expression of tryptophan hydroxylase-1 (TPH1) in duodenum and colon (N = 39), and (iv) 5-HT content in primary EC cells from these regions (N = 85). RESULTS: Plasma 5-HT was twofold higher in obese than control responders prior to (P = 0.025), and during (iAUC, P = 0.009), intraduodenal glucose infusion, and related positively to BMI (R2 = 0.334, P = 0.003) and HbA1c (R2 = 0.508, P = 0.009). The density of EC cells in the duodenum was twofold higher at baseline in obese subjects than controls (P = 0.023), with twofold more EC cells activated by glucose infusion in the obese (EC cells co-expressing 5-HT and pERK, P = 0.001), while the 5-HT content of EC cells in duodenum and colon was similar; TPH1 expression was 1.4-fold higher in the duodenum of obese subjects (P = 0.044), and related positively to BMI (R2 = 0.310, P = 0.031). CONCLUSIONS: Human obesity is characterized by an increased capacity to produce and release 5-HT from the proximal small intestine, which is strongly linked to higher body mass, and glycemic control. Gut-derived 5-HT is likely to be an important driver of pathogenesis in human obesity and dysglycemia.

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.

Neurally mediated propagating discrete clustered contractions superimposed on myogenic ripples in ex vivo segments of human ileum.

Narrow muscle strips have been extensively used to study intestinal contractility. Larger specimens from laboratory animals have provided detailed understanding of mechanisms that underlie patterned intestinal motility. Despite progress in animal tissue, investigations of motor patterns in large, intact specimens of human gut ex vivo have been sparse. In this study, we tested whether neurally dependent motor patterns could be detected in isolated specimens of intact human ileum. Specimens (n = 14; 7-30 cm long) of terminal ileum were obtained with prior informed consent from patients undergoing colonic surgery for removal of carcinomas. Preparations were set up in an organ bath with an array of force transducers, a fiberoptic manometry catheter, and a video camera. Spontaneous and distension-evoked motor activity was recorded, and the effects of lidocaine, which inhibits neural activity, were studied. Myogenic contractions (ripples) occurred in all preparations (6.17 ± 0.36/min). They were of low amplitude and formed complex patterns by colliding and propagating in both directions along the specimen at anterograde velocities of 4.1 ± 0.3 mm/s and retrogradely at 4.9 ± 0.6 mm/s. In five specimens, larger amplitude clusters of contractions were seen (discrete clustered contractions), which propagated aborally at 1.05 ± 0.13 mm/s and orally at 1.07 ± 0.09 mm/s. These consisted of two to eight phasic contractions that aligned with ripples. These motor patterns were abolished by addition of lidocaine (0.3 mM). The ripples continued unchanged in the presence of this neural blocking agent. These results demonstrate that both myogenic and neurogenic motor patterns can be studied in isolated specimens of human small intestine.

A panel of genes methylated with high frequency in colorectal cancer.

BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

From the organ bath to the whole person: a review of human colonic motility.

Motor function of the colon is essential for health. Our current understanding of the mechanisms that underlie colonic motility are based upon a range of experimental techniques, including molecular biology, single cell studies, recordings from muscle strips, analysis of part or whole organ ex vivo through to in vivo human recordings. For the surgeon involved in the clinical management of colonic conditions this amounts to a formidable volume of material. Here, we synthesize the key findings from these various experimental approaches so that surgeons can be better armed to deal with the complexities of the colon.

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.

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.

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.

Alterations in GLP-1 and PYY release with aging and body mass in the human gut.

The lining of our intestinal surface contains an array of hormone-producing cells that are collectively our bodies' largest endocrine cell reservoir. These "enteroendocrine" (EE) cells reside amongst the billions of absorptive epithelial and other cell types that line our gastrointestinal tract and can sense and respond to the ever-changing internal environment in our gut. EE cells release an array of important signalling molecules that can act as hormones, including glucagon-like peptide (GLP-1) and peptide YY (PYY) which are co-secreted from L cells. While much is known about the effects of these hormones on metabolism, insulin secretion and food intake, less is understood about their secretion from human intestinal tissue. In this study we assess whether GLP-1 and PYY release differs across human small and large intestinal tissue locations within the gastrointestinal tract, and/or by sex, body weight and the age of an individual. We identify that the release of both hormones is greater in more distal regions of the human colon, but is not different between sexes. We observe a negative correlation of GLP-1 and BMI in the small, but not large, intestine. Increased aging correlates with declining secretion of both GLP-1 and PYY in human large, but not small, intestine. When the data for large intestine is isolated by region, this relationship with age remains significant for GLP-1 in the ascending and descending colon and in the descending colon for PYY. This is the first demonstration that site-specific differences in GLP-1 and PYY release occur in human gut, as do site-specific relationships of L cell secretion with aging and body mass.

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.

Characterization of motor patterns in isolated human colon: are there differences in patients with slow-transit constipation?

The patterns of motor activity that exist in isolated full-length human colon have not been described. Our aim was to characterize the spontaneous motor patterns in isolated human colon and determine whether these patterns are different in whole colons obtained from patients with slow-transit constipation (STC). The entire colon (excluding the anus), was removed from patients with confirmed STC and mounted longitudinally in an organ bath ∼120 cm in length, containing oxygenated Krebs' solution at 36°C. Changes in circular muscle tension were recorded from multiple sites simultaneously along the length of colon, by use of isometric force transducers. Recordings from isolated colons from non-STC patients revealed cyclical colonic motor complexes (CMCs) in 11 of 17 colons, with a mean interval and half-duration of contractions of 4.0 ± 0.6 min and 51.5 ± 15 s, respectively. In the remaining six colons, spontaneous irregular phasic contractions occurred without CMCs. Interestingly, in STC patients robust CMCs were still recorded, although their CMC pacemaker frequencies were slower. Intraluminal balloon distension of the ascending or descending colon evoked an ascending excitatory reflex contraction, or evoked CMC, in 8 of 30 trials from non-STC (control) colons, but not from colons obtained from STC patients. In many control segments of descending colon, spontaneous CMCs consisted of simultaneous ascending excitatory and descending inhibitory phases. In summary, CMCs can be recorded from isolated human colon, in vitro, but their intrinsic pacemaker frequency is considerably faster in vitro compared with previous human recordings of CMCs in vivo. The observation that CMCs occur in whole colons removed from STC patients suggests that the intrinsic pacemaker mechanisms underlying their generation and propagation are preserved in vitro, despite impaired transit along these same regions in vivo.

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 effects of loperamide on excitatory and inhibitory neuromuscular function in the human colon.

BACKGROUND: In most animal species, opioids alter colonic motility via the inhibition of excitatory enteric motor neurons. The mechanisms by which opioids alter human colonic motility are unclear. The aim of this study was to describe the effects of loperamide on neuromuscular function in the human colon. METHODS: Tissue specimens of human colon from 10 patients undergoing an anterior resection were divided into three inter-taenial circular muscle strips. Separate organ baths were used to assess: (1) excitatory transmission (selective blockade of inhibitory transmission: L-NOARG/MRS2179); (2) inhibitory transmission (selective blockade of excitatory transmission: hyoscine hydrobromide); and (3) a control bath (no drug additions). Neuromuscular function was assessed using force transducer recordings and electrical field stimulation (EFS; 20 V, 10 Hz, 0.5 ms, 10 s) prior to and following loperamide and naloxone. KEY RESULTS: In human preparations with L-NOARG/MRS2179, loperamide had no significant effects on isometric contractions. In preparations with hyoscine hydrobromide, loperamide reduced isometric relaxation during EFS (median difference + 0.60 g post-loperamide, Z = -2.35, p = 0.019). CONCLUSIONS AND INFERENCES: Loperamide had no effect on excitatory neuromuscular function in human colonic circular muscle. These findings suggest that loperamide alters colonic function by acting primarily on inhibitory motor neurons, premotor enteric neurons, or via alternative non-opioid receptor pathways.

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.