Altered gastrointestinal motility involving autoantibodies in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that, in addition to motor, sensory, and cognitive symptoms, also causes constipation, which is poorly understood. Here, we characterize gastrointestinal (GI) dysmotility in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and evaluate whether autoantibodies target the enteric nervous system (ENS) and cause dysmotility. METHODS: EAE was induced in male SJL and B6 mice. GI motility was assessed in vivo and ex vivo in wild type (WT) and B cell-deficient mice. MS and EAE serum was used to survey potential targets in the ENS and changes in the ENS structure were characterized using immunohistochemistry. KEY RESULTS: EAE mice developed accelerated gastric emptying and delayed whole GI transit with reduced colonic motility. Fecal water content was reduced, and colonic migrating myoelectrical complexes (CMMC) and slow waves were less frequent. Colons from EAE mice exhibited decreased GFAP levels in glia. Sera from MS patients and from EAE mice targeted ENS neurons and glia. B-cell deficiency in EAE protected against colonic dysmotility. CONCLUSIONS & INFERENCES: Consistent with symptoms experienced in MS, we demonstrate that EAE mice widely exhibit features of GI dysmotility that persisted in the absence of extrinsic innervation, suggesting direct involvement of ENS neurocircuitry. The absence of GI dysmotility in B cell-deficient mice with EAE together with EAE and MS serum immunoreactivity against ENS targets suggests that MS could be classified among other diseases known to induce autoimmune GI dysmotility.

Extensive projections of myenteric serotonergic neurons suggest they comprise the central processing unit in the colon.

BACKGROUND: 5-Hydroxytryptamine (5-HT, serotonin) is an important regulator of colonic motility and secretion; yet the role of serotonergic neurons in the colon is controversial. METHODS: We used immunohistochemical techniques to examine their projections throughout the enteric nervous system and interstitial cells of Cajal (ICC) networks in the murine proximal to mid colon. KEY RESULTS: Serotonergic neurons, which were mainly calbindin positive, occurred only in myenteric ganglia (1 per 3 ganglia). They were larger than nNOS neurons but similar in size to Dogiel Type II (AH) neurons. 5-HT neurons, appeared to make numerous varicose contacts with each other, most nNOS neurons, Dogiel Type II/AH neurons and glial cells. 5-HT, calbindin and nNOS nerve fibers also formed a thin perimuscular nerve plexus that was associated with ganglia, which contained both nNOS positive and negative neurons, which lay directly upon the submucosal pacemaker ICC network. Neurons in perimuscular ganglia were surrounded by 5-HT varicosities. Submucous ganglia contained nNOS positive and negative neurons, and calbindin positive neurons, which also appeared richly supplied by serotonergic nerve varicosities. Serotonergic nerve fibers ran along submucosal arterioles, but not veins. Varicosities of serotonergic nerve fibers were closely associated with pacemaker ICC networks and with intramuscular ICC (ICC-IM). 5-HT2B receptors were found on a subpopulation of non-5-HT containing myenteric neurons and their varicosities, pacemaker ICC-MY and ICC-IM. CONCLUSIONS & INFERENCES: Myenteric serotonergic neurons, whose axons exhibit considerable divergence, regulate the entire enteric nervous system and are important in coordinating motility with secretion. They are not just interneurons, as regularly assumed, but possibly also motor neurons to ICC and blood vessels, and some may even be sensory neurons.

Red chlorophyll: the new barium?

Imaging of gastrointestinal (GI) motility remotely through the abdominal wall has always been a tradeoff between resolution and invasiveness. Skin reflects and/or absorbs wavelengths of radiation in the ultraviolet and visible ranges, but is largely transparent to both high-energy radiation (Gamma to X-rays; <0.1-10 nm) and low-energy radiation (infrared to radio waves; 700 nm-10 m). Imaging using short wavelength radiation such as X-ray cinematography has excellent spatial and temporal resolution, but ionization can produce acute and long-term deleterious effects to the patient or animal. Other 'slice-based' imaging techniques such as ultrasound/MRI/CT minimize tissue damage, but are limited in the planar area that can be imaged in a timely fashion. This viewpoint article will summarize and explore the implications of recent advances in infrared imaging of the GI tract, in particular, an article published in this issue of NGM entitled 'In vivo dynamic imaging of intestinal motions using diet-related autofluorescence' in which the authors have used infrared imaging in combination with that most elusive ingredient, standard mouse chow, to capture the motions of the mouse GI tract.

Activity in varicosities within the myenteric plexus between and during the colonic migrating motor complex in the isolated murine large intestine.

BACKGROUND: Neuronal communication within the myenteric plexus occurs when action potentials along nerve fibers produce Ca(2+) transients in varicosities leading to exocytosis of vesicles and neurotransmitters release. We used Ca(2+) transients in varicosities to monitor action potential activity in myenteric nerve pathways both between and during the colonic migrating motor complex (CMMC) in the isolated murine colon. METHODS: Strips of longitudinal muscle were removed to reveal the myenteric ganglia, which were then loaded with Fluo-4. KEY RESULTS: Many varicosities, including synaptotagmin 1 labeled varicosities, exhibited ongoing Ca(2+) transients (duration of unitary Ca(2+) transient 3.9 s). Between CMMCs, varicosities fired at a frequency of 0.6 Hz, which correlated with spontaneous inhibitory junction potentials in the circular muscle, suggesting they were mainly in inhibitory nerve pathways. During a CMMC other previously quiescent varicosities fired at 1.3 Hz (max. 2.0 Hz) for the duration (24 s) of the CMMC, suggesting they were on excitatory nerve pathways. Activity in varicosities was correlated with Ca(2+) transient responses in a number of neurons. Some varicosities appeared to release an inhibitory neurotransmitter that reduced activity in nNOS-positive neurons. Varicosities along the same nerve fiber exhibited identical patterns of activity that allowed nerve fibers to be traced throughout the myenteric plexus and internodal strands. Activity in varicosities was reduced by hexamethonium (100 µmol L(-1) ), and blocked by ω-conotoxin GVIA (200 nM) and tetrodotoxin (1 µmol L(-1) ; TTX). CONCLUSIONS & INFERENCES: Ca(2+) imaging of varicosities allows for a determination of activity in neural pathways within the enteric nervous system.

Movement based artifacts may contaminate extracellular electrical recordings from GI muscles.

BACKGROUND: Electrical slow waves drive peristaltic contractions in the stomach and facilitate gastric emptying. In gastroparesis and other disorders associated with altered gastric emptying, motility defects have been related to altered slow wave frequency and disordered propagation. Experimental and clinical measurements of slow waves are made with extracellular or abdominal surface recording. METHODS: We tested the consequences of muscle contractions and movement on biopotentials recorded from murine gastric muscles with array electrodes and pairs of silver electrodes. KEY RESULTS: Propagating biopotentials were readily recorded from gastric sheets composed of the entire murine stomach. The biopotentials were completely blocked by nifedipine (2 µmol L(-1) ) that blocked contractile movements and peristaltic contractions. Wortmannin, an inhibitor of myosin light chain kinase, also blocked contractions and biopotentials. Stimulation of muscles with carbachol increased the frequency of biopotentials in control conditions but failed to elicit biopotentials with nifedipine or wortmannin present. Intracellular recording with microelectrodes showed that authentic gastric slow waves occur at a faster frequency typically than biopotentials recorded with extracellular electrodes, and electrical slow waves recorded with intracellular electrodes were unaffected by suppression of movement. Electrical transients, equal in amplitude to biopotentials recorded with extracellular electrodes, were induced by movements produced by small transient stretches (<1 mm) of paralyzed or formalin fixed gastric sheets. CONCLUSIONS & INFERENCES: These data demonstrate significant movement artifacts in extracellular recordings of biopotentials from murine gastric muscles and suggest that movement suppression should be an obligatory control when monitoring electrical activity and characterizing propagation and coordination of electrical events with extracellular recording techniques.

Interstitial cells of Cajal in the cynomolgus monkey rectoanal region and their relationship to sympathetic and nitrergic nerves.

The morphology of interstitial cells of Cajal (ICC) in the circular muscle layer of the cynomolgus monkey internal anal sphincter (IAS) and rectum and their relationship to sympathetic and nitrergic nerves were compared by dual-labeling immunohistochemistry. Contractile studies confirmed that nitrergic nerves participate in neural inhibition in both regions whereas sympathetic nerves serve as excitatory motor nerves only in the IAS. Muscle bundles extended from myenteric to submucosal edge in rectum but in the IAS bundles were further divided into "minibundles" each surrounded by connective tissue. Dual labeling of KIT and smooth muscle myosin revealed KIT-positive stellate-shaped ICC (ICC-IAS) within each minibundle. In the rectum intramuscular ICC (ICC-IM) were spindle shaped whereas stellate-shaped ICC were located at the myenteric surface (ICC-MY). ICC were absent from both the myenteric and submucosal surfaces of the IAS. Nitrergic nerves (identified with anti-neuronal nitric oxide synthase antibodies or NADPH diaphorase activity) and sympathetic nerves (identified with anti-tyrosine hydroxylase antibody) each formed a plexus at the myenteric surface of the rectum but not the IAS. Intramuscular neuronal nitric oxide synthase- and tyrosine hydroxylase-positive fibers were present in both regions but were only closely associated with ICC-IM in rectum. Minimal association was also noted between ICC-IAS and cells expressing the nonspecific neuronal marker PGP9.5. In conclusion, the morphology of rectal ICC-IM and ICC-MY is similar to that described elsewhere in the gastrointestinal tract whereas ICC-IAS are unique. The distribution of stellate-shaped ICC-IAS throughout the musculature and their absence from both the myenteric and submucosal surfaces suggest that ICC-IAS may serve as pacemaker cells in this muscle whereas their limited relationship to nerves suggests that they are not involved in neuromuscular transmission. Additionally, the presence of numerous minibundles, each containing both ICC-IAS and nerves, suggests that this muscle functions as a multiunit type muscle.

ICC-MY coordinate smooth muscle electrical and mechanical activity in the murine small intestine.

BACKGROUND: Animals carrying genetic mutations have provided powerful insights into the role of interstitial cells of Cajal (ICC) in motility. One classic model is the W/W(V) mouse which carries loss-of-function mutations in c-kit alleles, but retains minimal function of the tyrosine kinase. Previous studies have documented loss of slow waves and aberrant motility in the small intestine of W/W(V) mice where myenteric ICC (ICC-MY) are significantly depleted. METHODS: Here, we used morphological and electrophysiological techniques to further assess the loss of ICC around the circumference of the small intestine and determine consequences of losing ICC-MY on electrical activity, Ca(2+) transients and contractions of the longitudinal muscle (LM). KEY RESULTS: In wild-type mice, there was coherent propagation of Ca(2+) transients through the ICC-MY network and spread of this activity to the LM. In short segments of small intestine in vitro and in exteriorized segments, slow waves coordinated smoothly propagating Ca(2+) waves and contractions in the LM of wild-type mice. In W/W(V) mice, Ca(2+) waves were initiated at variable sites along and around intestinal segments and propagated without constraint unless they collided with other Ca(2+) waves. This activity resulted in abrupt, uncoordinated contractions. CONCLUSIONS & INFERENCES: These results show how dominance of pacemaking by ICC-MY coordinates propagating con-tractions and regulates the spontaneous activity of smooth muscle.

Non-peristaltic patterns of motor activity in the guinea-pig proximal colon.

BACKGROUND: The guinea-pig proximal colon contains semi-solid feces which are propelled by intermittent neural peristaltic waves to the distal colon, where solid pellets are formed. Between propulsive periods, complex motor patterns underlie fluid re-absorption and mixing of contents. METHODS: Spatio-temporal analysis of video recordings were used to investigate neural and myogenic patterns of non-peristaltic motor activity. KEY RESULTS: At low distension (6 cmH(2)O), two major motor patterns were seen. Narrow rings of constriction (abrupt contractions) occurred at 19 cpm. These previously undescribed contractions occurred, almost simultaneously, at many points along the preparation, with a calculated propagation velocity of 110 mm s(-1). They were abolished by hexamethonium and by tetrodotoxin, indicating they were neurally mediated. Inhibition of nitric oxide synthase resulted in increased frequency of 'abrupt contractions' suggesting ongoing inhibitory modulation by endogenous nitric oxide. After tetrodotoxin, another distinct motor pattern was revealed; 'ripples'(1) consisted of shallow rings of contraction, occurring at 18 cpm and propagating at 2.7-2.9 mm s(-1) orally or aborally from multiple initiation sites. The frequency of 'ripples' increased as intraluminal pressure was raised, becoming very irregular at high distensions. L-type calcium channel blockers and openers affected the amplitude of 'ripples'. No frequency gradient of 'ripples' along the proximal colon was detected. This absence explains the multiple initiation sites which often shifted over time, and the oral and aboral propagation of 'ripples'. CONCLUSIONS & INFERENCES: The interaction of myogenic 'ripples' with neurogenic 'abrupt contractions' generates localized alternating rings of contractions and dilatation, well suited to effective mixing of contents.

CaM kinase II in colonic smooth muscle contributes to dysmotility in murine DSS-colitis.

BACKGROUND: Altered calcium mobilization has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The aim of this study was to investigate the mechanisms by which disrupted intracellular Ca(2+) signalling contributes to the impaired contractility of colon circular smooth muscles. METHODS: Acute colitis was induced in C57Bl/6 mice with dextran sulphate sodium (DSS) in the drinking water for 5 days. KEY RESULTS: Spontaneous and acetylcholine-evoked contractions, caffeine-evoked hyperpolarization, and SERCA2 and phospholamban expression were reduced compared with controls. Tetrodotoxin did not restore control levels of contractile activity. The amplitudes, but not the frequency, of intracellular Ca(2+) waves were increased compared with controls. Caffeine abolished intracellular Ca(2+) waves in control smooth muscle cells, but not in smooth muscle cells from DSS-treated mice. CaM kinase II activity and cytosolic levels of HDAC4 were increased, and I kappaB alpha levels were decreased in distal colon smooth muscles from DSS-treated mice. CONCLUSIONS & INFERENCES: These results suggest that disruptions in intracellular Ca(2+) mobilization due to down-regulation of SERCA2 and phospholamban expression lead to increased CaM kinase II activity and cytosolic HDAC4 that may contribute to the dysmotility of colonic smooth muscles in colitis by enhancing NF-kappaB activity.

Identification of capsaicin-sensitive rectal mechanoreceptors activated by rectal distension in mice.

Rodents detect visceral pain in response to noxious levels of rectal distension. However, the mechanoreceptors that innervate the rectum and respond to noxious levels of rectal distension have not been identified. Here, we have identified the mechanoreceptors of capsaicin-sensitive rectal afferents and characterized their properties in response to circumferential stretch of the rectal wall. We have also used the lethal spotted (ls/ls) mouse to determine whether rectal mechanoreceptors that respond to capsaicin and stretch may also develop in an aganglionic rectum that is congenitally devoid of enteric ganglia. In wild type (C57BL/6) mice, graded increases in circumferential stretch applied to isolated rectal segments activated a graded increase in firing of slowly-adapting rectal mechanoreceptors. Identical stimuli applied to the aganglionic rectum of ls/ls mice also activated similar graded increases in firing of stretch-sensitive rectal afferents. In both wild type and aganglionic rectal preparations, focal compression of the serosal surface using von Frey hairs identified mechanosensitive "hot spots," that were associated with brief bursts of action potentials. Spritzing capsaicin (10 microM) selectively onto each identified mechanosensitive hot spot activated an all or none discharge of action potentials in 32 of 56 identified hot spots in wild type mice and 24 of 62 mechanosensitive hot spots in the aganglionic rectum of ls/ls mice. Each single unit activated by both capsaicin and circumferential stretch responded to low mechanical thresholds (1-2 g stretch). No high threshold rectal afferents were ever recorded in response to circumferential stretch. Anterograde labeling from recorded rectal afferents revealed two populations of capsaicin-sensitive mechanoreceptor that responded to stretch: one population terminated within myenteric ganglia, the other within the circular and longitudinal smooth muscle layers. In the aganglionic rectum of ls/ls mice, only the i.m. mechanoreceptors were identified. Both myenteric and i.m. mechanoreceptors could be identified by their immunoreactivity to the anti-TRPV1 antibody and the vesicular glutamate transporter, Vglut2. Myenteric mechanoreceptors had a unique morphology, consisting of smooth bulbous nodules that ramified within myenteric ganglia. In summary, the rectum of wild type mice is innervated by at least two populations of capsaicin-sensitive rectal mechanoreceptor, both of which respond to low mechanical thresholds within the innocuous range. These findings suggest that the visceral pain pathway activated by rectal distension is likely to involve low threshold rectal mechanoreceptors that are activated within the normal physiological range.

Methionine and its derivatives increase bladder excitability by inhibiting stretch-dependent K(+) channels.

BACKGROUND AND PURPOSE: During the bladder filling phase, the volume of the urinary bladder increases dramatically, with only minimal increases in intravesical pressure. To accomplish this, the smooth muscle of the bladder wall must remain relaxed during bladder filling. However, the mechanisms responsible for the stabilization of bladder excitability during stretch are unclear. We hypothesized that stretch-dependent K(+) (TREK) channels in bladder smooth muscle cells may inhibit contraction in response to stretch. EXPERIMENTAL APPROACHES: Bladder tissues from mouse, guinea pig and monkey were used for molecular, patch clamp, mechanical, electrical, Ca(2+) imaging and cystometric responses to methionine and its derivatives, which are putative blockers of stretch-dependent K(+) (SDK) channels. KEY RESULTS: SDK channels are functionally expressed in bladder myocytes. The single channel conductance of SDK channels is 89pS in symmetrical K(+) conditions and is blocked by L-methionine. Expressed TREK-1 currents are also inhibited by L-methioninol. All three types of bladder smooth muscle cells from mouse, guinea pig and monkey expressed TREK-1 genes. L-methionine, methioninol and methionine methyl ester but not D-methionine increased contractility in concentration-dependent manner. Methioninol further increased contractility and depolarized the membrane in the presence of blockers of Ca(2+)-activated K(+) conductance. L-methionine induced Ca(2+) waves that spread long distances through the tissue under stretched conditions and were associated with strong contractions. In cystometric assays, methioninol injection increased bladder excitability mimicking overactive bladder activity. CONCLUSIONS AND IMPLICATIONS: Methioninol-sensitive K(+) (SDK, TREK-1) channels appear to be important to prevent spread of excitation through the syncitium during bladder filling.

Recent advances in enteric neurobiology: mechanosensitive interneurons.

Until recently, it was generally assumed that the only intrinsic sensory neuron, or primary afferent neuron, in the gut was the after-hyperpolarizing AH/Type II neuron. AH neurons excited by local chemical and mechanical stimulation of the mucosa appear to be necessary for activating the peristaltic reflex (oral excitation and anal inhibition of the muscle layers) and anally propagating ring like contractions (peristaltic waves) that depend upon smooth muscle tone. However, our recent findings in the guinea-pig distal colon suggest that different neurochemical classes of interneuron in the colon are also mechanosensitive in that they respond directly to changes in muscle length, rather than muscle tone or tension. These interneurons have electrophysiological properties consistent with myenteric S-neurons. Ascending and descending interneurons respond directly to circumferential stretch by generating an ongoing polarized peristaltic reflex activity (oral excitatory and anal inhibitory junction potentials) in the muscle for as long as the stimulus is maintained. Some descending (nitric oxide synthase +ve) interneurons, on the other hand, appear to respond directly to longitudinal stretch and are involved in accommodation and slow transit of faecal pellets down the colon. This review will present recent evidence that suggests some myenteric S interneurons, in addition to AH neurons, behave as intrinsic sensory neurons.

Propagation of pacemaker activity in the guinea-pig antrum.

Cyclical periods of depolarization (slow waves) underlie peristaltic contractions involved in mixing and emptying of contents in the gastric antrum. Slow waves originate from a myenteric network of interstitial cells of Cajal (ICC-MY). In this study we have visualized the sequence and propagation of Ca(2+) transients associated with pacemaker potentials in the ICC network and longitudinal (LM) and circular muscle (CM) layers of the isolated guinea-pig gastric antrum. Gastric antrum was dissected to reveal the ICC-MY network, loaded with Fluo-4 AM and activity was monitored at 37 degrees C. Ca(2+) waves propagated throughout the ICC-MY network at an average velocity of 3.24 +/- 0.12 mm s(-1) at a frequency of 4.87 +/- 0.16 cycles min(-1) (n= 4). The propagation of the Ca(2+) wave often appeared 'step-like', with separate regions of the network being activated after variable delays. The direction of propagation was highly variable (Delta angle of propagation 44.3 +/- 10.9 deg per cycle) and was not confined to the axes of the longitudinal or circular muscle. Ca(2+) waves appeared to spread out radially from the site of initiation. The initiating Ca(2+) wave in ICC-MY was correlated to secondary Ca(2+) waves in intramuscular interstitial cells of Cajal, ICC-IM, and smooth muscle cells, and the local distortion (contraction) in a field of view. TTX (1 microm) had little effect on slow wave or pacemaker potential activity, but 2-APB (50 microm) blocked all Ca(2+) waves, indicating a pivotal role for intracellular Ca(2+) stores. Nicardipine (2 microm) eliminated the Ca(2+) transient generated by smooth muscle, but did not affect the fast upstroke associated with ICC-MY. These results indicate that slow waves follow a sequence of activation, beginning with the ICC-MY and ICC-IM network, followed later by a sustained Ca(2+) transient in the muscle layers that is responsible for contraction.

Analysis of motor patterns in the isolated guinea-pig large intestine by spatio-temporal maps.

We investigated and quantified the spontaneous patterns of motility in the isolated guinea-pig proximal and distal colon taken from adult animals. During spontaneous emptying, profiles of proximal and distal colon were recorded with a video camera, and image analysis was used to construct spatio-temporal maps of the motions of the intestinal wall. Four patterns of motility were recorded. In the proximal colon there were neurally mediated contractions that propagated in the aboral direction at 4.1 mm s(-1), gently pushing the soft contents aborally; these are likely to represent spontaneous peristaltic behaviour. A second pattern, insensitive to tetrodotoxin (TTX; 0.6 microM), consisted, in both oral and aboral propagation, of shallow contractions of the circular muscle (ripples). These contractions propagated aborally at 2.8 +/- 0.45 mm s(-1) and orally at 2.03 +/- 0.31 mm s(-1) (n=10). Of these TTX-resistant contractions, 22.5% propagated both orally and aborally from a common origin. The orally propagated component of these myogenic contractions is likely to correspond to the antiperistalsis widely described in the proximal colon. In the distal colon, two patterns of motor activity were observed. One, induced by natural or artificial pellets, consisted of peristaltic contractions that pushed the pellets aborally at 0.8 mm s(-1) and expelled a pellet every 108 s. In the interval between pellet propulsion and after the distal colon had emptied all of its pellets a second, nerve-mediated pattern of motor activity, consisting of clusters of annular circular muscle contractions separated by short dilated regions, slowly propagated aborally at 0.3 mm s(-1). Both of these motor patterns were abolished by TTX (0.6 microM). A latex balloon, inserted at the oral end of the empty isolated distal colon and inflated to a size similar to faecal pellets, was propelled at 1.4 mm s(-1). Epoxy resin-covered natural pellets were propelled at a similar speed of 1.6 mm s(-1). Our data revealed that myogenic and neurogenic patterns of propagated contractions in the colon occur in isolated preparations and are involved in emptying the colon.

Spatial and temporal coordination of junction potentials in circular muscle of guinea-pig distal colon.

1. In isolated, stretched, flat-sheet preparations of guinea-pig distal colon, simultaneous intracellular recordings were made from pairs of circular muscle (CM) cells to map the region of smooth muscle at which spontaneous junction potentials (sJPs) were coordinated in both space and time. 2. Spontaneous inhibitory junction potentials (sIJPs) and excitatory junction potentials (sEJPs) were recorded from all animals with varying frequencies and amplitudes (up to 25 mV). 3. Large amplitude (> or = 9 mV) sIJPs or sEJPs with near-identical amplitudes and time courses were recorded synchronously from two CM cells, even when the two electrodes were separated by up to 11 mm in the circumferential axis and < or = 4 mm in the longitudinal axis. However, smaller (< 9 mV) sIJPs or sEJPs were less coordinated and exhibited greater variability in their times to peak. 4. The standard deviation (S.D.) for the time difference between the peaks of sJPs was related to the amplitude of the events and the distance between the electrodes. The S.D. for large amplitude JPs (approximately 30 ms), which was less than that for small JPs (approximately 150 ms), remained constant across the circumferential axis (at least up to 6 mm), but declined rapidly for distances > or = 2 mm in the longitudinal axis. 5. Current injection (up to 8 nA) into a single CM cell elicited electrotonic potentials in neighbouring CM cells, only when the two electrodes were separated by less than 100 microm circumferentially. Beyond 50 microm electronic potentials were rarely detected. 6. Tetrodotoxin (TTX; 1 microM) abolished all sJPs, whereas hexamethonium (300 microM) either abolished, or substantially reduced all sJPs. 7. Nitro-L-arginine (L-NA; 100 microM) abolished the slow repolarisation phase of sIJPs without any apparent effect on the amplitude of sIJPs. Apamin abolished the fast, initial component of sIJPs, suggesting synchronous release of two inhibitory neurotransmitters during the sIJP. Atropine (1 microM) abolished sEJPs. 8. No sJPs were recorded from the CM layer when it was separated from the myenteric plexus. 9. In conclusion, sIJPs and sEJPs in colonic CM occur synchronously over large regions of the smooth muscle syncitium. The results are discussed in relation to the idea that spontaneous junction potentials in colonic CM are not monoquantal events, but are generated by the simultaneous release of transmitter from many release sites, due to the synchronous activation of many enteric motor neurons.

Quantitative analysis of peristalsis in the guinea-pig small intestine using spatio-temporal maps.

1. Peristalsis was evoked in guinea-pig small intestine by slow fluid infusion and recorded onto video and digitized. Spatio-temporal maps of diameter and longitudinal movement were constructed and parameters of motion were calculated. 2. During the filling of the isolated segments of intestine, rhythmic local longitudinal movements were observed at several points along the preparation. These phasic longitudinal muscle contractions were associated with small but significant local increases in diameter and probably reflect a passive mechanical coupling by connective tissue in the gut wall. In addition, occasional synchronized longitudinal muscle contractions caused net shortening of the preparation and always preceded the onset of peristaltic emptying. 3. Peristaltic emptying was characterized by a contraction of the circular muscle which usually started at the oral end of the preparation, that propagated aborally, propelling the contents. However, in 19 % of trials, the first circular muscle contraction occurred in the aboral half of the preparation. 4. The propagation of peristalsis consisted of separate sequential circular muscle contractions several centimetres long, particularly in the oral half of the preparation, giving a 'step-like' appearance to the spatio-temporal map. The gut was transiently distended aboral to the propagating circular muscle contraction due to the propulsion of contents. 5. At each point in the preparation, the longitudinal muscle remained contracted during the propulsive part of the circular muscle contraction. Only when the circular muscle contraction became lumen occlusive did lengthening of the longitudinal muscle take place. 6. Spatio-temporal maps are a powerful tool to visualize and analyse the complexity of gastrointestinal motility patterns.

Rapid anterograde and retrograde tracing from mesenteric nerve trunks to the guinea-pig small intestine in vitro.

A novel technique for rapid anterograde labelling of cut axons in vitro was used to visualise the peripheral branches of mesenteric nerve trunks supplying the guinea-pig small intestine. Biotinamide, dissolved in an artificial intracellular solution, was applied to the cut ends of the mesenteric nerves and the tissue was maintained in organ culture overnight. Labelled nerve fibres were visualised by fluorescein isothiocyanate (FITC)-conjugated streptavidin. Intense staining of nerve fibres and terminal varicosities in the ganglia and internodal strands of the myenteric plexus was achieved up to 15 mm from the application site. Filled fibres formed baskets around some myenteric nerve cell bodies, suggesting target-specific neurotransmission. When combined with multiple-labelling immunohistochemistry for tyrosine hydroxylase (TH), calcitonin gene-related protein (CGRP) or choline acetyltransferase (ChAT), most anterogradely labelled nerve fibres, and many pericellular baskets, were found to be TH immunoreactive, indicating their postganglionic sympathetic origin. Double-labelling immunohistochemistry revealed that the postganglionic sympathetic pericellular baskets preferentially surrounded 5-hydroxytryptamine (5-HT)-handling myenteric neurons. Some biotinamide-filled fibres were CGRP immunoreactive, and are likely to originate from spinal sensory neurons. We describe for the first time many pericellular baskets labelled from the mesenteric nerves which were ChAT immunoreactive. Retrogradely filled intestinofugal nerve cell bodies were also observed, all of which had a single axon arising from a small nerve cell body with short filamentous or lamellar dendrites. Many of these cells were ChAT immunoreactive. This in vitro technique is effective in identifying the fine arrangement of nerve terminals arising from nerve trunks in the periphery.

The projections of 5-hydroxytryptamine-accumulating neurones in the myenteric plexus of the small intestine of the guinea-pig.

Retrograde tracing, combined with immunohistochemistry, was used to study the projections of 5-hydroxytryptamine (5-HT)-accumulating neurones within the ileum of the guinea-pig, with confocal microscopy being used to characterise further their morphology. Two classes of neurones in the myenteric plexus, capable of taking up 5-HT or analogues, were distinguished. One class had Dogiel type I morphology with lamellar dendrites, was located on the edge or in the middle of ganglia and lacked immunoreactivity for somatostatin (SOM). The other class had smooth ovoid cell bodies with multiple filamentous dendrites and a single axon and represented a subset of the SOM-immunoreactive interneurones in the myenteric plexus. Varicosities immunoreactive for 5-HT alone, 5-HT/SOM or SOM alone were present in the myenteric ganglia. Both classes of 5-HT-accumulating neurones had long aboral projections within the myenteric plexus (up to 100 mm long) and to the submucous plexus and probably function as descending interneurones.