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We have investigated the tone dependence of the intrinsic nervous activity generated by localized wall distension in isolated segments of guinea-pig distal colon using mechanical recordings and video imaging of wall movements. A segment of colon was threaded through two partitions, which divided the colon for pharmacological purposes into oral, stimulation and anal regions. An intraluminal balloon was located in the stimulation region between the two partitions (12 mm apart). Maintained colonic distension by an intraluminal balloon or an artificial faecal pellet held at a fixed location generated rhythmic (frequency 0.3 contractions min(-1); duration approximately 60 s) peristaltic waves of contraction. Video imaging of colonic wall movements or the selective application of pharmacological agents suggested that peristaltic waves originated just oral (< or = 4 mm) to the pellet and propagated both orally (approximately 11 mm s(-1)) and anally (approximately 1 mm s(-1)). Also, during a peristaltic wave the colon appears to passively shorten in front of a pellet, as a result of an active contraction of the longitudinal muscle oral to the pellet. Faecal pellet movement only occurred when a rhythmic peristaltic wave was generated. Rhythmic peristaltic waves were abolished in all regions by the smooth muscle relaxants isoproterenol (1 microM), nicardipine (1 microM) or papavarine (10 microM), and by the neural antagonists tetrodotoxin (TTX; 0.6 microM), hexamethonium (100 microM) or atropine (1 microM), when added selectively to the stimulation region. Nicardipine, atropine, TTX, or hexamethonium (100 microM) also blocked the evoked peristaltic waves when selectively added to the oral region. Nomega-nitro-L-arginine (L-NA; 100 microM) added to the anal region reduced the anal relaxation but increased the anal contraction, leading to an increase in the apparent conduction velocity of each peristaltic wave. In conclusion, maintained distension by a fixed artificial pellet generates propulsive, rhythmic peristaltic waves, whose enteric neural activity is critically dependent upon smooth muscle tone. These peristaltic waves usually originate just oral to the pellet, and their apparent conduction velocity is generated by activation of descending inhibitory nerve pathways.

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells (<200 microm apart) at the oral region of the colon showed that excitatory junction potentials (EJPs) discharged synchronously in both muscle layers for periods of up to 6 h. Similarly, at the anal region of the colon, inhibitory junction potentials (IJPs) discharged synchronously in the two muscle layers. Quadruple recordings from LM and CM orally at the same time as from the LM and CM anally revealed that IJPs occurred synchronously in the LM and CM anally at the same time as EJPs in LM and CM located 20 mm orally. Oral EJPs and anal IJPs were linearly related in amplitude between the two muscle layers. Spatiotemporal maps generated from simultaneous video imaging of the movements of the colon, combined with intracellular recordings, revealed that some LM contractions orally could be correlated in time with IJPs in CM cells anally. N(omega)-nitro-L-arginine (L-NA; 100 microM) abolished the IJP in LM, whereas a prominent L-NA-resistant "fast" IJP was always observed in CM. In summary, in stretched preparations, synchronized EJPs in both LM and CM orally are generated by synchronized firing of many ascending interneurons, which simultaneously activate excitatory motor neurons to both muscle layers. Similarly, synchronized IJPs in both LM and CM anally are generated by synchronized firing of many descending interneurons, which simultaneously activate inhibitory motor neurons to both muscle layers. This synchronized motor activity ensures that both muscles around the entire circumference are excited orally at the same time as inhibited anally, thus producing net aboral propulsion.

Simultaneous intracellular recordings were made from pairs of circular muscle (CM) cells, at the oral and anal ends of a segment of guinea-pig distal colon, to investigate the neuronal mechanisms underlying faecal pellet propulsion. When a minimum degree of circumferential stretch was applied to sheet preparations of colon, recordings from CM cells revealed either no ongoing junction potentials, or alternatively, small potentials usually < 5 mV in amplitude. Maintained circumferential stretch applied to these preparations evoked an ongoing discharge of excitatory junction potentials (EJPs) at the oral recording site (range: 1-25 mV), which lasted for up to 6 h. The onset of each large oral EJP was time-locked with the onset of an inhibitory junction potential (IJP) at an anal recording electrode, located 2 cm from the oral recording. Similar results were obtained in isolated intact tube preparations of colon, when recordings were made immediately oral and anal of an artificial faecal pellet. The amplitudes of many large (> 5 mV) oral EJPs were linearly related to the amplitudes of anal IJPs occurring 20 mm apart. In the absence of an L-type Ca(2+) channel blocker, action potentials occurred on each large oral EJP. Synchronized discharges of stretch-activated EJPs and IJPs were preserved following pretreatment with capsaicin (10 microM), were unaffected by nifedipine (1 microM) and did not require the mucosa or submucous plexus. EJPs and IJPs were abolished by hexamethonium (300 microM) or tetrodotoxin (1 microM), but persisted in the presence of pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 microM) or an NK(3) tachykinin receptor antagonist (Neurokinin A 4-10; 100 nM to 5 microM). In summary, maintained circumferential stretch of the distal colon activates a population of intrinsic mechanosensory neurons that generate repetitive firing of ascending excitatory and descending inhibitory pathways to CM. These mechanosensory neurons, which may be interneurons, are stretch sensitive, rather than muscle tension sensitive, since they are resistant to muscular paralysis. We suggest the synchrony in onset of oral EJPs and anal IJPs over large regions of colon is due to synchronous synaptic activation of ascending and descending interneurons.

Ca2+ wave activity was monitored in the longitudinal (LM) layer of isolated murine caecum and proximal colon at 35 degrees C with fluo-4 AM and an iCCD camera. Both intracellular (within LM cells) and intercellular (also spreading from cell to cell) Ca2+ waves were observed. Intracellular Ca2+ waves were associated with a lack of muscle movement whereas intercellular Ca2+ waves, which were five times more intense than intracellular waves, were often associated with localized contractions. Several intracellular Ca2+ waves were present at the same time in individual LM cells. Waves in adjacent LM cells were not coordinated and were unaffected by TTX (1 microM) but were blocked by IP3 receptor antagonists xestospongin-C (Xe-C; 2 microM) or 2-aminoethyl diphenylborate (2-APB; 25 microM), and by ryanodine (10 microM). Caffeine (5 mM) restored wave activity following blockade with Xe-C. NiCl2 (1 mM) blocked intracellular Ca2+ waves, and nicardipine (2 microM) reduced their frequency and intensity, but did not affect their velocity, suggesting the sarcoplasmic reticulum may be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves often occurred in bursts and propagated rapidly across sizeable regions of the LM layer and were blocked by heptanol (0.5 mM). Intercellular Ca2+ waves were dependent upon neural activity, external Ca2+ entry through L-type Ca2+ channels, and amplification via calcium-induced calcium release (CICR). In conclusion, intracellular Ca2+ waves, which may reduce muscle excitability, are confined to individual LM cells. They depend upon Ca2+ release from internal Ca2+ stores and are likely to be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves, which are likely to underlie smooth muscle tone, mixing and propulsion, depend upon neural activity, muscle action potential propagation and amplification by CICR.

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 microm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 microM) or tetrodotoxin (1 microM) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 microM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca(2+) channels.

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.

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.

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.

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.

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.

1. We have described and analysed the movements of the isolated stomach during distension by correlating intragastric pressure with video recordings, and investigated the presence of intrinsic inhibitory and excitatory reflexes. 2. Isolated guinea-pig stomachs, placed in an organ bath, were slowly distended with Krebs solution using a syringe pump via a cannula through the pylorus. The changes in intragastric pressure during cycles of distension were monitored by pressure transducers connected to both oesophageal and pyloric cannulae. The resistivity of the gastric wall (change in pressure with volume, delta P/delta V) and the amplitude and frequency of phasic pressure events were calculated from pressure recordings. 3. The movements of the stomach were also recorded onto videotape. The motion of the gastric wall during distension cycles was analysed to establish the patterns of contractions, their propagation and the distribution of fluid in the stomach. During filling, fluid was preferentially accommodated in the fundus. Propagating (peristaltic) contractions, often starting in the fundus, moved aborally towards the pylorus. The peak of the phasic pressure event was observed when a contraction reached the orad antrum. As it reached the pylorus, intragastric pressure was at its minimum. 4. During the initial phase of distension, intragastric pressure increased steeply. Tetrodotoxin and hyoscine reduced both the resistivity and amplitude of phasic pressure events. Hexamethonium had a similar effect. Thus distension appears to activate an excitatory reflex pathway, involving nicotinic ganglionic transmission. This reflex increases wall tension and enhances myogenic peristaltic contractions. 5. In control preparations, with larger distension volumes, the intragastric pressure decreased, despite the continued infusion of Krebs solution. L-NAME and apamin abolished this drop in pressure, indicating that gastric enteric inhibitory mechanisms prevail at larger distension volumes. After blockade of the excitatory reflex, hexamethonium antagonized the inhibitory response, indicating that activation of inhibitory mechanisms involves nicotinic transmission, probably on enteric inhibitory motoneurons. 6. Both the excitatory and inhibitory reflexes in the isolated stomach operate within a physiological range of gastric volumes. The excitatory reflex predominates at small distension volumes, leading to large phasic propagated contractions that mix the contents and may lead to emptying of the stomach. The inhibitory reflex, described previously as adaptive relaxation, can maximally relax the stomach and is activated preferentially at higher distension volumes to accommodate the contents. The interplay of these reflex pathways in the isolated stomach produces a rich repertoire of gastric movements. 7. The isolated stomach preparation, used with a combination of kinematic, kinetic and pharmacological methods, provides a highly suitable means of investigating the mechanisms of gastric motility.