The enteric nervous system (ENS) consists of thousands of small ganglia arranged in the submucosal and myenteric plexuses, which can be negatively affected by Crohn's disease and ulcerative colitis - inflammatory bowel diseases (IBDs). IBDs are complex and multifactorial disorders characterized by chronic and recurrent inflammation of the intestine, and the symptoms of IBDs may include abdominal pain, diarrhea, rectal bleeding, and weight loss. The P2X7 receptor has become a promising therapeutic target for IBDs, especially owing to its wide expression and, in the case of other purinergic receptors, in both human and model animal enteric cells. However, little is known about the actual involvement between the activation of the P2X7 receptor and the cascade of subsequent events and how all these activities associated with chemical signals interfere with the functionality of the affected or treated intestine. In this review, an integrated view is provided, correlating the structural organization of the ENS and the effects of IBDs, focusing on cellular constituents and how therapeutic approaches through the P2X7 receptor can assist in both protection from damage and tissue preservation.

Glia in the central nervous system exert precise spatial and temporal regulation over neural circuitry on a synapse-specific basis, but it is unclear if peripheral glia share this exquisite capacity to sense and modulate circuit activity. In the enteric nervous system (ENS), glia control gastrointestinal motility through bidirectional communication with surrounding neurons. We combined glial chemogenetics with genetically encoded calcium indicators expressed in enteric neurons and glia to study network-level activity in the intact myenteric plexus of the proximal colon. Stimulation of neural fiber tracts projecting in aboral, oral, and circumferential directions activated distinct populations of enteric glia. The majority of glia responded to both oral and aboral stimulation and circumferential pathways, while smaller subpopulations were activated only by ascending and descending pathways. Cholinergic signaling functionally specifies glia to the descending circuitry, and this network plays an important role in repressing the activity of descending neural pathways, with some degree of cross-inhibition imposed upon the ascending pathway. Glial recruitment by purinergic signaling functions to enhance activity within ascending circuit pathways and constrain activity within descending networks. Pharmacological manipulation of glial purinergic and cholinergic signaling differentially altered neuronal responses in these circuits in a sex-dependent manner. Collectively, our findings establish that the balance between purinergic and cholinergic signaling may differentially control specific circuit activity through selective signaling between networks of enteric neurons and glia. Thus, enteric glia regulate the ENS circuitry in a network-specific manner, providing profound insights into the functional breadth and versatility of peripheral glia.

Here, we recognise some of the extraordinary accomplishments of the partnership between Geoff Burnstock and Mollie Holman, and the everlasting impact they both made in autonomic neuroscience in Australia. Much of strength today in autonomic neuroscience can be traced back to a time when Geoff and Mollie commenced their seminal studies on autonomic neuroscience, initially at Oxford, then at The University of Melbourne in the mid 1960's. Mollie and Geoff published their first paper together, at Oxford, with their then mentor, and doyenne of smooth muscle, Professor Edith Bülbring. They did not always agree on the interpretation of their own scientific findings. Geoff was convinced early on that Adenosine triphosphate (ATP), or a related purine, was an excitatory neurotransmitter at peripheral sympathetic neuroeffector junctions. Mollie was reticent for decades. However, she began to take the notion seriously that ATP maybe a neurotransmitter, when receptors for purines were identified in the 1990's. What the partnership between Mollie and Geoff taught us in Australia was to not fear respectful criticism, but rather to be receptive to and embrace objective, collegial and constructive scientific peer-review. One of the many great legacies of Geoff and Mollie was the large number of researchers, who were fortunate disciples of their supervision, and who have now themselves gone on to make significant discoveries in autonomic and visceral neuroscience. This review summarizes some of their major legacies and represents a very personal historical perspective of the two authors, pupils respectively of Mollie and Geoff.

This is a very personal reminiscence of the long period of Enteric Nervous System research in which I have been involved. I started to work on the gut in the early 60s really because in Turin when I arrived from Argentina, where my family migrated temporarily after the WWII, nobody was seriously working on the brain. In Anatomy they were studying the neural "intramural plexuses" and that for me was close enough to the nervous system. I grew up in the mountains near Turin near the French border where our ex-family house still bears our name. I joined the Department of Anatomy as an intern student and I was privileged to seat at a desk where a previous generation of young scientists, who studied under the professor of Anatomy A. Levi, the founder of the methods for culturing neural tissue. They were Salvador Luria, Renato Dulbecco and Rita Levi-Montalcini, who, after migrating to the USA, were each were given the Noble prize.

1. The enteric nervous system modulates intestinal behaviours, such as motor patterns and secretion. Although much is known about different types of neurons and simple reflexes in the intestine, it remains unclear how complex behaviours are generated. 2. Mathematical modelling is an important tool for assisting the understanding of how the neurons and reflexes can be pieced together to generate intestinal behaviours. 3. Models have identified a functional role for slow excitatory post-synaptic potentials (EPSPs) by distinguishing between fast and slow EPSPs in the ascending excitation reflex. These models also discovered coordinated firing of similarly located neurons as emergent properties of feed-forward networks of interneurons in the intestine. A model of the recurrent network of intrinsic sensory neurons identified important control mechanisms to prevent uncontrolled firing due to positive feedback and that the interaction between these control mechanisms and slow EPSPs is necessary for the networks to encode ongoing sensory stimuli. This model also showed that such networks may mediate migrating motor complexes. 4. A network model of vasoactive intestinal peptide neurons in the submucosal plexus found this relatively sparse recurrent network could produce uncontrolled firing under conditions that appear to be related to cholera toxin-induced hypersecretion. 5. Abstract modelling of the intestinal fed-state motor patterns has identified how stationary contractions can arise from a polarized network. 6. These models have also helped predict and/or explained pharmacological evidence for two rhythm generators and the requirement of feedback from contractions in the circular muscle.

Accumulating evidence demonstrates that acetylcholine can directly modulate immune function in peripheral tissues including the spleen and gastrointestinal tract. However, the anatomical relationships between the peripheral cholinergic system and immune cells located in these lymphoid tissues remain unclear due to inherent technical difficulties with currently available neuroanatomical methods. In this study, mice with specific expression of the tdTomato fluorescent protein in choline acetyltransferase (ChAT)-expressing cells were used to label preganglionic and postganglionic cholinergic neurons and their projections to lymphoid tissues. Notably, our anatomical observations revealed an abundant innervation in the intestinal lamina propria of the entire gastrointestinal tract principally originating from cholinergic enteric neurons. The aforementioned innervation frequently approached macrophages, plasma cells, and lymphocytes located in the lamina propria and, to a lesser extent, lymphocytes in the interfollicular areas of Peyer's patches. In addition to the above innervation, we observed labeled epithelial cells in the gallbladder and lower intestines, as well as Microfold cells and T-cells within Peyer's patches. In contrast, we found only a sparse innervation in the spleen consisting of neuronal fibers of spinal origin present around arterioles and in lymphocyte-containing areas of the white pulp. Lastly, a small population of ChAT-expressing lymphocytes was identified in the spleen including both T- and B-cells. In summary, this study describes the variety of cholinergic neuronal and nonneuronal cells in a position to modulate gastrointestinal and splenic immunity in the mouse.

IGLEs represent the only low-threshold vagal mechanosensory terminals in the tunica muscularis of the esophagus. Previously, close relationships of vesicular glutamate transporter 2 (VGLUT2) immunopositive IGLEs and cholinergic varicosities suggestive for direct contacts were described in almost all mouse esophageal myenteric ganglia. Possible cholinergic influence on IGLEs requires specific acetylcholine receptors. In particular, the occurrence and location of neuronal muscarinic acetylcholine receptors (mAChR) in the esophagus were not yet characterized.

This study aimed at specifying relationships of VGLUT2 immunopositive IGLEs and vesicular acetylcholine transporter (VAChT)-immunopositive varicosities using pre-embedding electron microscopy and the location of mAChR1-3 (M1-3) within esophagus and nodose ganglia using multilabel immunofluorescence and retrograde tracing.

Electron microscopy confirmed synaptic contacts between cholinergic varicosities and IGLEs. M1- and M2-immunoreactivities (-iry; -iries) were colocalized with VGLUT2-iry in subpopulations of IGLEs. Retrograde Fast Blue tracing from the esophagus showed nodose ganglion neurons colocalizing tracer and M2-iry. M1-3-iries were detected in about 80% of myenteric ganglia and in about 67% of myenteric neurons. M1- and M2-iry were present in many fibers and varicosities within myenteric ganglia. Presynaptic M2-iry was detected in all, presynaptic M3-iry in one-fifth of motor endplates of striated esophageal muscles. M1-iry could not be detected in motor endplates of the esophagus, but in sternomastoid muscle.

Acetylcholine probably released from varicosities of both extrinsic and intrinsic origin may influence a subpopulation of esophageal IGLEs via M2 and M1-receptors.

Hyoscine butylbromide (HBB, Buscopan(®) ) is clinically used to treat intestinal cramps and visceral pain. Various studies, mainly on animal tissues, suggested that its antimuscarinic action is responsible for its spasmolytic effect. However, functional in vitro studies with human tissue have not been performed so far.

We wanted to provide a comprehensive study on the mode of action of HBB in human intestinal samples and investigated HBB (1 nmol L(-1) -10 μmol L(-1)) effects on muscle activity with isometric force transducers and calcium imaging, on epithelial secretion with Ussing chamber technique and on enteric neurons using fast neuroimaging.

Hyoscine butylbromide concentration dependently reduced muscle contractions, calcium mobilization, and epithelial secretion induced by the muscarinic agonist bethanechol with IC50 values of 429, 121, and 224 nmol L(-1), respectively. Forskolin-induced secretion was not altered by HBB. Cholinergic muscarinic muscle and epithelial responses evoked by electrical nerve stimulation were inhibited by 1-10 μmol L(-1) HBB. Moreover, HBB significantly reduced the bethanechol-induced action potential discharge in enteric neurons. Interestingly, we observed that high concentrations of HBB (10 μmol L(-1)) moderately decreased nicotinic receptor-mediated secretion, motility, and nerve activity.

The results demonstrated the strong antimuscarinic action of HBB whereas the nicotinic antagonism at higher concentrations plays at most a moderate modulatory role. The muscle relaxing effect of HBB and its inhibition of muscarinic nerve activation likely explain its clinical use as an antispasmodic drug. Our results further highlight a so far unknown antisecretory action of HBB which warrants further clinical studies on its use in secretory disorders.

Transcripts of the choline acetyltransferase (ChAT) gene reveal a number of different splice variants including ChAT of a peripheral type (pChAT). Immunohistochemical staining of the brain using an antibody against pChAT clearly revealed peripheral cholinergic neurons, but failed to detect cholinergic neurons in the central nervous system. In rodents, pChAT-immunoreactivity has been detected in cholinergic parasympathetic postganglionic and enteric ganglion neurons. In addition, pChAT has been observed in non-cholinergic neurons such as peripheral sensory neurons in the trigeminal and dorsal root ganglia. The common type of ChAT (cChAT) has been investigated in many parts of the brain and the spinal cord of non-human primates, but little information is available about the localization of pChAT in primate species. Here, we report the detection of pChAT immunoreactivity in trigeminal ganglion (TG) neurons and its co-localization with Substance P (SP) and/or calcitonin gene-related peptide (CGRP) in the cynomolgus monkey, Macaca fascicularis. Neurons positive for pChAT were observed in a rather uniform pattern in approximately half of the trigeminal neurons throughout the TG. Most pChAT-positive neurons had small or medium-sized cell bodies. Double-immunofluorescence staining showed that 85.1% of SP-positive cells and 74.0% of CGRP-positive cells exhibited pChAT immunoreactivity. Most pChAT-positive cells were part of a larger population of neurons that co-expressed SP and/or CGRP.

After a meal, the gastrointestinal tract exhibits a set of behaviours known as the fed state. A major feature of the fed state is a little understood motor pattern known as segmentation, which is essential for digestion and nutrient absorption. Segmentation manifests as rhythmic local constrictions that do not propagate along the intestine. In guinea-pig jejunum in vitro segmentation constrictions occur in short bursts together with other motor patterns in episodes of activity lasting 40-60 s and separated by quiescent episodes lasting 40-200 s. This activity is induced by luminal nutrients and abolished by blocking activity in the enteric nervous system (ENS). We investigated the enteric circuits that regulate segmentation focusing on a central feature of the ENS: a recurrent excitatory network of intrinsic sensory neurons (ISNs) which are characterized by prolonged after-hyperpolarizing potentials (AHPs) following their action potentials. We first examined the effects of depressing AHPs with blockers of the underlying channels (TRAM-34 and clotrimazole) on motor patterns induced in guinea-pig jejunum, in vitro, by luminal decanoic acid. Contractile episode durations increased markedly, but the frequency and number of constrictions within segmenting bursts and quiescent period durations were unaffected. We used these observations to develop a computational model of activity in ISNs, excitatory and inhibitory motor neurons and the muscle. The model predicted that: i) feedback to ISNs from contractions in the circular muscle is required to produce alternating activity and quiescence with the right durations; ii) transmission from ISNs to excitatory motor neurons is via fast excitatory synaptic potentials (EPSPs) and to inhibitory motor neurons via slow EPSPs. We conclude that two rhythm generators regulate segmentation: one drives contractions within segmentation bursts, the other the occurrence of bursts. The latter depends on AHPs in ISNs and feedback to these neurons from contraction of the circular muscle.

The peripheral type of choline acetyltransferase (pChAT) is an isoform of the well-studied common type of choline acetyltransferase (cChAT), the synthesizing enzyme of acetylcholine. Since pChAT arises by exons skipping, its amino acid sequence is similar to that of cChAT, except the lack of a continuous peptide sequence encoded by all the four exons from 6 to 9. While cChAT expression has been observed in both the central and peripheral nervous systems, pChAT is preferentially expressed in the peripheral nervous system. pChAT appears to be a reliable marker for the visualization of peripheral cholinergic neurons and their processes, whereas other conventional markers including cChAT have not been used successfully for it. In mammals like rodents, pChAT immunoreactivity has been observed in most, if not all, physiologically identified peripheral cholinergic structures such as all parasympathetic postganglionic neurons and most neurons of the enteric nervous system. In addition, pChAT has been found in many peripheral neurons that are derived from the neural crest. These include sensory neurons of the trigeminal ganglion and the dorsal root ganglion, and sympathetic postganglionic neurons. Recent studies moreover indicate that pChAT, as well as cChAT, appears ubiquitously expressed among various species not only of vertebrate mammals but also of invertebrate mollusks. This finding implies that the alternative splicing mechanism to generate pChAT and cChAT has been preserved during evolution, probably for some functional benefits.

Digestion and absorption of nutrients and the secretion and reabsorption of fluid in the gastrointestinal tract are regulated by neurons of the enteric nervous system (ENS), the extensive peripheral nerve network contained within the intestinal wall. The ENS is an important physiological model for the study of neural networks since it is both complex and accessible. At least 20 different neurochemically and functionally distinct classes of enteric neurons have been identified in the guinea pig ileum. These neurons express a wide range of ionotropic and metabotropic receptors. Synaptic potentials mediated by ionotropic receptors such as the nicotinic acetylcholine receptor, P2X purinoceptors and 5-HT(3) receptors are seen in many enteric neurons. However, prominent synaptic potentials mediated by metabotropic receptors, like the P2Y(1) receptor and the NK(1) receptor, are also seen in these neurons. Studies of synaptic transmission between the different neuron classes within the enteric neural pathways have shown that both ionotropic and metabotropic synaptic potentials play major roles at distinct synapses within simple reflex pathways. However, there are still functional synapses at which no known transmitter or receptor has been identified. This review describes the identified roles for both ionotropic and metabotropic neurotransmission at functionally defined synapses within the guinea pig ileum ENS. It is concluded that metabotropic synaptic potentials act as primary transmitters at some synapses. It is suggested identification of the interactions between different synaptic potentials in the production of complex behaviours will require the use of well validated computer models of the enteric neural circuitry.

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300-900 ms) or slow (durations 1.3-9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2-4-disulfonic acid (30 microM, N = 3) or the specific P2Y(1) antagonist MRS 2179 (10 microM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 (N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y(1), S neurons).

Nociceptin is the endogenous agonist of the "orphan" opioid receptor-1 (ORL-1). We investigated whether activation of the ORL-1 receptor influences smooth muscle contractility and enteric neurotransmission within ascending myenteric reflex pathways of rats.

Reverse transcriptase polymerase chain reaction was performed to evaluate the presence of ORL-1 receptors. The ascending part of the ascending myenteric reflex in rats was studied in ileal segments using a 3-chambered organ bath. Intracellular recordings were performed to evaluate pharmacologic effects on excitatory and inhibitory junction potentials (EJP; IJP). Single- and double-labeling immunohistochemistry was used to examine the distribution of ORL-1 within the intestinal wall.

ORL-1 expression and immunoreactivity was found in the large majority of myenteric neurons. In addition to the cholinergic myenteric neurons, all nitrergic myenteric neurons expressed the ORL-1 receptor. Nociceptin significantly reduced cholinergic twitch contractions, an effect that was reversed by the ORL-1 receptor antagonist [Nphe(1)]nociceptin(1-13)NH(2). Neither nociceptin nor [Nphe(1)]nociceptin(1-13)NH(2) had a direct influence on smooth muscle contractility. Nociceptin significantly reduced ascending myenteric reflex contractions and prolonged the latency from stimulation to contraction. Both effects were antagonized by [Nphe(1)]nociceptin(1-13)NH(2). Intracellular recordings demonstrated that nociceptin reduces the cholinergically mediated EJP and the nitrergic phase of IJP in a concentration-dependent manner, effects that were reversible in presence of [Nphe(1)]nociceptin(1-13)NH(2).

We conclude that activation of ORL-1 receptors on myenteric neurons reduce excitatory and inhibitory neurotransmission within the gastrointestinal tract. This is accompanied by a reduction of the small intestinal peristaltic reflex response. These effects might be used pharmacologically.

This study examined whether mucosal stimulation activates long secretomotor neural reflexes and, if so, how they are organized. The submucosa of in vitro full thickness guinea pig ileal preparations was exposed in the distal portion and intracellular recordings were obtained from electrophysiologically identified secretomotor neurons. Axons in the intact mucosa of the oral segment were stimulated by a large bipolar stimulating electrode. In control preparations, a single stimulus pulse evoked a fast excitatory postsynaptic potential (EPSP) in 86% of neurons located 0.7-1.0 cm anal to the stimulus site. A stimulus train evoked multiple fast EPSPs, but slow EPSPs were not observed. To examine whether mucosal stimulation specifically activated mucosal sensory nerve terminals, the mucosa/submucosa was severed from the underlying layers and repositioned. In these preparations, fast EPSPs could not be elicited in 89% of cells. Superfusion with phorbol dibutyrate enhanced excitability of sensory neurons and pressure-pulse application of serotonin to the mucosa increased the fast EPSPs evoked by mucosal stimulation, providing further evidence that sensory neurons were involved. To determine whether these reflexes projected through the myenteric plexus, this plexus was surgically lesioned between the stimulus site and the impaled neuron. No fast EPSPs were recorded in these preparations following mucosal stimulation whereas lesioning the submucosal plexus had no effect. These results demonstrate that mucosal stimulation triggers a long myenteric pathway that activates submucosal secretomotor neurons. This pathway projects in parallel with motor and vasodilator reflexes, and this common pathway may enable coordination of intestinal secretion, blood flow, and motility.

The physiology and pathophysiology of the network of interstitial cells of Cajal associated with the deep muscular plexus (ICC-DMP) of the small intestine are still poorly understood. The objectives of the present study were to evaluate the effects of inflammation associated with Trichinella spiralis infection on the ICC-DMP and to correlate loss of function with structural changes in these cells and associated structures. We used immunohistochemistry, electron microscopy, and assessment of distention-inducing electrophysiological parameters in vitro. Damage to ICC-DMP was associated with a loss of distention-induced patterns of electrical activity normally associated with distention-induced peristalsis. Consistently, the timing of recovery of ICC-DMP paralleled the timing of recovery of the distention-induced activity. Nerve varicosities associated with ICC-DMP including cholinergic nerves, assessed by immunoelectron microscopy and whole mount double labeling, paralleled injury to ICC-DMP thus contributing to impaired excitatory innervation of smooth muscle cells. Major additional changes included a remodeling of the inner circular muscle layer, which may affect long-term sensitivity to distention after infection. In conclusion, transient injury to ICC-DMP in response to T. spiralis infection is severe and associated with a complete lack of distention-induced burst-type muscle activity.

Exogenously administered galanin inhibits cholinergic transmission to the longitudinal muscle and reduces peristaltic efficiency in the guinea pig ileum with a mechanism partially mediated by galanin receptor 1 (GAL-R1). We investigated the effect of exogenous galanin 1-16, which has high affinity for GAL-R1, on the ascending excitatory reflex of the circular muscle elicited by radial distension in isolated segments of guinea pig ileum. We used a three-compartment bath that allows dissecting the ascending pathway into the oral (site of excitatory motor neurons), intermediate (site of ascending interneurons) and caudal compartment (site of intrinsic primary afferent neurons). Galanin 1-16 (0.3-3 micromol L(-1)) applied to the oral compartment inhibited in a concentration-dependent manner the ascending excitatory reflex elicited by the wall distension in the caudal compartment. This effect was antagonized by the GAL-R1 antagonist, RWJ-57408 (1 and 10 micromol L(-1)). By contrast, galanin 1-16 was ineffective when added to the intermediate or caudal compartment up to 3 micromol L(-1). GAL-R1 immunoreactive neurons did not contain neuron-specific nuclear protein, a marker for intrinsic primary afferent neurons. These findings indicate that GAL-R1s are present on motor neurons responsible for the ascending excitatory reflex, but not on ascending interneurons and intrinsic primary afferent neurons.

We examined specific receptor/transmitter combinations used at functionally identified synapses in ascending and descending reflex pathways of guinea pig distal colon. Excitatory (EJPs) or inhibitory junction potentials (IJPs) were recorded intracellularly from nicardipine-paralyzed circular smooth muscle in either the oral or anal recording chamber of a three-chambered organ bath, respectively. Blockade of synaptic transmission in the central chamber with a 0.25 mM Ca2+/12 mM Mg2+ solution abolished EJPs evoked by distension applied either in the central or the far (anal) chamber. IJPs evoked by distension in the central or the far (oral) chamber were depressed to approximately 50% of control. Hexamethonium (nicotinic receptor antagonist, 200 microM) in the central chamber reduced IJPs evoked by far or central distension to 50%, whereas EJPs evoked by far distension were abolished and EJPs evoked by central distension were reduced to 70% of control. Hexamethonium in the recording chambers reduced both IJPs and EJPs evoked by central distension to approximately 50%. EJPs in the ascending pathway were unaffected by blockade of muscarinic receptors in the central chamber or blockade of neurokinin 3 tachykinin receptors in this or the recording chamber. In the descending pathway, blockade of P2 receptors in the same chambers had only a minor effect on distension-evoked IJPs. Thus some intrinsic sensory neurons of guinea pig colon have long descending projections (>30 mm), but ascending projections of <15 mm. In contrast to the ileum, transmission between ascending or descending interneurons and from sensory neurons to descending interneurons is predominantly via nicotinic receptors; but transmission to inhibitory or excitatory motoneurons and from sensory neurons to ascending interneurons involves nicotinic and other unidentified receptors.

Cerulein and neostigmine are prokinetic drugs whose potency and effective dose range are barely known. The aim of this study was to assess their benefit for normal and compromised peristalsis.

In vitro, isolated segments of guinea pig small intestine. Setting : University laboratory.

Small bowel segments were mounted in tissue baths and luminally perfused with Tyrode solution. Test drugs (prokinetic: cerulein, neostigmine; inhibitory: atropine, hexamethonium, epinephrine, sufentanil) were added to the tissue bath.

Peristalsis was quantified via changes in the peristaltic pressure threshold. One-way and two-way analysis of variance (ANOVA) were used for statistical analysis. Cerulein (0.03-100 nM) stimulated normal peristalsis in a concentration-dependent manner and reversed paralysis of peristalsis induced by all inhibitory test drugs to a similar extent. The properistaltic effect of neostigmine was limited to a narrow concentration range (0.03-0.1 micro M), whereas concentrations >0.3 micro M inhibited peristalsis. Neostigmine more effectively counteracted blockage of peristalsis caused by atropine than that caused by hexamethonium. The inhibitory effects of epinephrine and sufentanil on peristalsis were reversed only at the concentration range of 0.1-0.3 micro M neostigmine.

Cerulein stimulates normal peristalsis in vitro at a wide concentration range and reverses blockage of peristalsis caused by drugs with a site of action either on the enteric nervous system or intestinal smooth muscle. Neostigmine's prokinetic effect, to the contrary, is limited to a small concentration range and best seen when peristalsis is depressed by blockage of cholinergic muscle activation.

The mechanisms of prokinetic action of tegaserod in the gastrointestinal tract has not been studied in detail. The aim of this study was to investigate the effect of tegaserod on peristaltic reflexes and propagating peristaltic waves in guinea pig ileum. A partitioned organ bath divided into three chambers was used to investigate the effect of tegaserod on peristaltic reflexes. A sensory stimulus was applied to the intermediate chamber, and changes in the circular muscle tension were monitored in a peripheral chamber. Another peristaltic bath was used to investigate the effect of tegaserod on peristaltic waves induced by intraluminal perfusion. Guinea pig ileum exhibited contractions in the circular muscle both orally and anally in response to mucosal stroking. Tegaserod (10(-8) - 10(-6) M) did not influence the maximal amplitude and the area under the curve of contraction both orally and anally to a mucosal stimulus. Intraluminal perfusion of fluid containing tegaserod (10(-8) - 10(-6) M) significantly increased the number of peristaltic waves in a concentration-dependent manner (P<0.05). Also, tegaserod (10(-8) - 10(-6) M) significantly increased the area under the curve of peristaltic waves (P<0.05). It is concluded that tegaserod has prokinetic action on guinea pig ileum by increasing the number of the circular muscle contractions during peristalsis.

We examined descending reflex pathways in the rat colon using intracellular recording techniques. Inhibitory junction potentials (IJPs) were recorded from circular smooth muscle when descending pathways were excited by combined mucosal compression and distension. IJPs were reduced to 71% of control when synaptic transmission was blocked in the oral stimulation chamber of a divided organ bath suggesting that two reflex pathways exist, the one involving descending sensory neurones and the other involving descending interneurones. Hexamethonium (200 micromol L(-1)) in the recording chamber abolished reflexly evoked IJPs, while in the stimulation chamber, it was as effective as synaptic blockade. When hexamethonium was added to a chamber lying between the stimulation and recording chambers, it again sharply depressed IJPs to 27% of control; an extent similar to synaptic blockade. A P2 receptor antagonist did not reveal any purinergic neurotransmission. Either granisetron (5-HT3 receptor antagonist, 1 micromol L(-1)) or SB204070 (5-HT4 receptor antagonist, 1 micromol L(-1)) in the stimulation chamber significantly decreased IJPs; these decreases were not additive. We conclude that some sensory neurones and interneurones in rat colon have long anally projecting axons and that acetylcholine, acting via nicotinic receptors, is the primary neurotransmitter from sensory neurones, to inhibitory motor neurones and between interneurones.

The functional significance of the slow excitatory synaptic potentials (EPSPs) in myenteric neurones is unknown. We investigated this using intracellular recording from myenteric neurones in guinea-pig ileum, in vitro. In all, 121 neurones responded with fast EPSPs to distension of the intestine oral to the recording site. In 28 of these neurones, distension also evoked depolarizations similar to the slow EPSPs evoked by electrical stimulation in the same neurones. Intracellular injection of biocytin and immunohistochemistry revealed that neurones responding to distension with slow EPSPs were descending interneurones, which were immunoreactive for nitric oxide synthase (NOS). Other neurones, including inhibitory motor neurones and interneurones lacking NOS, did not respond to distension with slow EPSPs, but many had slow EPSPs evoked electrically. Slow EPSPs evoked electrically or by distension in NOS-immunoreactive descending interneurones were resistant to blockade of NK(1) or NK(3) tachykinin receptors (SR 140333, 100 nM; SR 142801, 100 nM, respectively) and group I metabotropic glutamate receptors (PHCCC, 10-30 microM), when the antagonists were applied in the recording chamber of a two-chambered organ bath. However, slow EPSPs evoked electrically in inhibitory motor neurones were substantially depressed by SR 140333 (100 nM). Blockade of synaptic transmission in the stimulation chamber of the organ bath abolished slow EPSPs evoked by distension, indicating that they arose from activity in interneurones, and not from anally directed, intrinsic sensory neurones. Thus, distension evokes slow EPSPs in a subset of myenteric neurones, which may be important for intestinal motility.

In the digestive tract, there is evidence for the presence of high amounts of endocannabinoids (anandamide and 2-arachidonylglycerol) and of mechanisms for endocannabinoid metabolism and possibly endocannabinoid uptake. Pharmacological studies have shown that anandamide inhibits excitatory transmission and peristalsis in the isolated guinea-pig ileum and reduces intestinal motility in the mouse in vivo; all these effects are mediated by CB(1) receptors, which are located on enteric nerves. Conversely, the selective CB(1) receptor antagonist SR141716A increased intestinal motility and this effect is likely due to the displacement of endocannabinoids rather than to its inverse agonist properties. Interestingly, inhibitory effects of anandamide via non-CB(1) receptors and stimulatory effects via vanilloid receptors have also been proposed.

1. Sympathetic regulation of the motility of guinea-pig ileum was investigated using mesenteric nerve (MN) stimulation to inhibit motility reflexes, in vitro. 2. Transmural electrical stimulation (5 Hz, 1 s) in intact intestinal segments, or inflation of a balloon against the mucosa in opened segments, evoked contractions of the circular and longitudinal muscles oral to the stimulus. 3. MN stimulation (10 Hz, 5 s) usually abolished contractions of the longitudinal and circular muscles evoked by either electrical or mechanical stimuli. 4. The inhibition was mimicked by UK14,304 (70-100 nM) and abolished by idazoxan (100 nM), revealing an enhancement of circular muscle contractions. There was no evidence for alpha(2)-receptors on the muscle, suggesting sympathetic inhibition was via the myenteric plexus. 5. Possible sites of action of noradrenaline released from sympathetic nerves were investigated using intracellular recordings from the circular muscle in a multichambered organ bath. 6. When in the stimulation chamber, UK14,304 depressed (by 50 %) excitatory junction potentials (EJPs) recorded oral to a distension stimulus, but did not affect inhibitory junction potentials (IJPs) recorded anal to the stimulus. When added to a chamber between the stimulus and recording chambers, UK14,304 depressed EJPs by 40 %, but did not alter IJPs. When in the recording chamber, UK14,304 depressed EJPs by 20 %, but had no effect on IJPs. IJPs were inhibited, however, when UK14,304 was applied to the whole bath. 7. It is concluded that sympathetic activity inhibits intestinal motility mainly via alpha(2)-adrenoceptors on ascending interneurons and intrinsic sensory neurons of the orally directed reflex pathway.

The pyloric sphincter (PS) controls gastric emptying and prevents the reflux of duodenal content into the stomach. Neuronal pathways and reflexes controlling the guinea-pig PS were physiologically investigated in isolated preparations. Simultaneous intracellular or extracellular and tension recordings from PS circular muscle with electrical and stretch stimulation were used. Electrical stimulation evoked an initial small contraction followed by a relaxation with a corresponding inhibitory junction potential (IJP) then a second large contraction with a corresponding excitatory junction potential (EJP). Hyoscine (1 micromol L-1) blocked the first contraction, and reduced the second contraction and EJP by 52.5% and 61%, respectively. These responses were further reduced by the NK2 antagonist, MEN10627 (1 micromol L-1), and the NK1 antagonist, SR140333 (1 micromol L-1). N-nitro-L-arginine (100 micro;mol L-1) and apamin (0.5 micromol L-1) blocked the relaxation and the IJP. Duodenal electrical stimulation evoked an EJP, whereas antral stimulation evoked an IJP followed by a small EJP. All were blocked by hexamethonium (100 micromol L-1). Duodenal stretch evoked tetrodotoxin-sensitive reflex contractions and membrane depolarization with action potentials in the PS. Thus, PS enteric motor neurones receive inputs from the duodenum and the stomach. There are stretch-sensitive ascending excitatory reflex pathways from the duodenum to the PS.

The role of NK3 receptors in rabbit colonic propulsion has been investigated in vitro with the selective agonist, senktide, and two selective antagonists, SR142801 and SB222200. Peristalsis was elicited by distending a rubber balloon with 0.3 and 1.0 mL of water leading to a velocity of 2.2 and 2.8 mm s-1, respectively. At concentrations of 1 nM, senktide inhibited propulsion evoked by both distensions (range 25-40%), whereas at 6 and 60 nmol L-1 facilitated 'submaximal' propulsion by 30%. In the presence of Nomega-nitro-L-arginine (L-NNA, 200 micromol L-1), which per se caused a slight prokinetic effect, 1 nmol L-1 senktide markedly accelerated propulsion (range 35-50%). Hexamethonium (200 micromol L-1) had minor effects on propulsion. In its presence, 60 nmol L-1 senktide significantly inhibited propulsion induced by both stimuli (range 20-50%). SR142801 (0.3, 3 nmol L-1) and SB222200 (30, 300 nmol L-1) facilitated 'submaximal' propulsion (range 20-40%). Conversely, higher antagonist concentrations (SR142801: 30, 300 nM; SB222200: 1, 10 micromol L-1) inhibited propulsion to both distensions by 20%. A combination of SR142801 (300 nmol L-1) plus hexamethonium (200 micromol L-1) induced an approximately four-fold greater inhibition of propulsion than that induced by SR142801 alone. In conclusion, in the rabbit-isolated distal colon, a subset of NK3 receptors located on descending pathways mediates an inhibitory effect on propulsion by activating a NO-dependent mechanism. Another subset of NK3 receptors, located on ascending pathways mediates a facilitative effect involving a synergistic interaction with cholinergic nicotinic receptors.

Gut motility in non-mammalian vertebrates as in mammals is controlled by the presence of food, by autonomic nerves and by hormones. Feeding and the presence of food initiates contractions of the stomach wall and subsequently gastric emptying, peristalsis, migrating motor complexes and other patterns of motility follow. This overview will give examples of similarities and differences in control systems between species. Gastric receptive relaxation occurs in fish and is an enteric reflex. Cholecystokinin reduces the rate of gastric emptying in fish as in mammals. Inhibitory control of peristalsis is exerted, e.g. by VIP, PACAP, NO in fish and amphibians, while excitatory stimuli arise from nerves releasing tachykinins, acetylcholine or serotonin (5-HT). In crocodiles, we have found the presence of the same nerve types, although the effects on peristalsis have not been studied. Recent studies on signal transduction in the gut smooth muscle of fish and amphibians suggest that external Ca2+ is of great importance, but not the only source of Ca2+ recruitment in tachykinin-, acetylcholine- or serotonin-induced contractions of rainbow trout and Xenopus gastrointestinal smooth muscle. The effect of acetylcholine involves reduction of cAMP-levels in the smooth muscle cells. It is concluded that, in general, the control systems in non-mammalian vertebrates are amazingly similar between species and animal groups and in comparison with mammals.

The role of P2X receptors in descending inhibitory reflexes evoked by distension or mucosal distortion in the guinea-pig ileum was studied using intracellular recording from the circular muscle in a two-chambered organ bath. This allowed separate superfusion of the sites of reflex stimulation and recording, thereby allowing drugs to be selectively applied to different parts of the reflex pathway. Inhibitory junction potentials (IJPs) evoked by electrical field stimulation (EFS) in the recording chamber were compared with those evoked during reflexes to control for effects of P2 receptor antagonists on neuromuscular transmission. The P2 receptor antagonists suramin (100 microM) and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (10 and 60 microM; PPADS), when added to the recording chamber, depressed reflexly evoked IJPs significantly more than those evoked by EFS. In particular, 10 microM PPADS depressed IJPs evoked by distension or mucosal distortion by about 50 %, but had little effect on IJPs evoked by EFS. Blockade of synaptic transmission in the stimulation chamber with a low Ca2+-high Mg2+ solution depressed, but did not abolish, IJPs evoked by distension. The residual reflex IJPs were unaffected by PPADS (10 microM), hyoscine (1 microM), hyoscine plus hexamethonium (200 microM), or hysocine plus hexamethonium plus PPADS in the recording chamber. We conclude that P2X receptors are important for synaptic transmission from descending interneurons to inhibitory motor neurons in descending inhibitory reflex pathways of guinea-pig ileum. Transmission from anally directed axons of distension-sensitive intrinsic sensory neurons to inhibitory motor neurons is unlikely to involve P2X, muscarinic or nicotinic receptors.

This study examined whether myenteric neurons activate submucosal vasodilator pathways in in vitro combined submucosal-myenteric plexus preparations from guinea pig ileum. Exposed myenteric ganglia were electrically stimulated, and changes in the outside diameter of submucosal arterioles were monitored in adjoining tissue by videomicroscopy. Stimulation up to 18 mm from the recording site evoked large TTX-sensitive vasodilations in both orad and aborad directions. In double-chamber baths, which isolated the stimulating myenteric chamber from the recording submucosal chamber, hexamethonium or the muscarinic antagonist 4-diphenylacetoxy-N-(2-chloroethyl)-piperdine hydrochloride (4-DAMP) almost completely blocked dilations when superfused in the submucosal chamber. When hexamethonium was placed in the myenteric chamber approximately 50% of responses were hexamethonium sensitive in both orad and aboard orientations. The addition of 4-DAMP or substitution of Ca(2+)-free, 12 mM Mg(2+) solution did not cause further inhibition. These results demonstrate that polysynaptic pathways in the myenteric plexus projecting orad and aborad can activate submucosal vasodilator neurons. These pathways could coordinate intestinal blood flow and motility.

This paper, written for the symposium in honour of more than 40 years' contribution to autonomic research by Professor Geoffrey Burnstock, highlights the progress made in understanding the organisation of the enteric nervous system over this time. Forty years ago, the prevailing view was that the neurons within the gut wall were post-ganglionic neurons of parasympathetic pathways. This view was replaced as evidence accrued that the neurons are part of the enteric nervous system and are involved in reflex and integrative activities that can occur even in the absence of neuronal influence from extrinsic sources. Work in Burnstock's laboratory led to the discovery of intrinsic inhibitory neurons with then novel pharmacology of transmission, and precipitated investigation of neuron types in the enteric nervous system. All the types of neurons in the enteric nervous system of the small intestine of the guinea-pig have now been identified in terms of their morphologies, projections, primary neurotransmitters and physiological identification. In this region there are 14 functionally defined neuron types, each with a characteristic combination of morphological, neurochemical and biophysical properties. The nerve circuits underlying effects on motility, blood flow and secretion that are mediated through the enteric nervous system are constructed from these neurons. The circuits for simple motility reflexes are now known, and progress has been made in analysing those involved in local control of blood flow and transmucosal fluid movement in the small intestine.

This study examined synaptic inputs from myenteric neurons innervating submucosal neurons. Intracellular recordings were obtained from submucosal S neurons in guinea pig ileal preparations in vitro, and synaptic inputs were recorded in response to electrical stimulation of exposed myenteric plexus. Most S neurons received synaptic inputs [>80% fast (f) excitatory postsynaptic potentials (EPSP), >30% slow (s) EPSPs] from the myenteric plexus. Synaptic potentials were recorded significant distances aboral (fEPSPs, 25 mm; sEPSPs, 10 mm) but not oral to the stimulating site. When preparations were studied in a double-chamber bath that chemically isolated the stimulating "myenteric chamber" from the recording side "submucosal chamber," all fEPSPs were blocked by hexamethonium in the submucosal chamber, but not by a combination of nicotinic, purinergic, and 5-hydroxytryptamine-3 receptor antagonists in the myenteric chamber. In 15% of cells, a stimulus train elicited prolonged bursts of fEPSPs (>30 s duration) that were blocked by hexamethonium. These findings suggest that most submucosal S neurons receive synaptic inputs from predominantly anally projecting myenteric neurons. These inputs are poised to coordinate intestinal motility and secretion.

1. We present evidence that adenosine triphosphate (ATP) plays a major role in excitatory neuro-neuronal transmission in ascending and descending reflex pathways to the longitudinal (LM) and circular muscle (CM). 2. A partitioned bath was used for the pharmacological isolation of a segment of guinea-pig ileum ( approximately 6 cm in length), allowing drugs to be selectively applied to an intermediate region between the region where mucosal stimulation was applied and that where mechanical recordings were made. 3. Brush stroking the mucosa (3 strokes) elicited a synchronous contraction of the LM and CM both above (ascending excitation) and below (descending excitation) the site of stimulation. All reflexes were abolished when tetrodotoxin (1 microM) was applied to the intermediate chamber. 4. Hexamethonium (300 microM) added to the intermediate chamber abolished the ascending contraction in 15 % of oral preparations (from 26 preparations, 18 animals) and the descending contraction in 13% of anal preparations studied (from 53 preparations, 48 animals). In the remaining 85% of oral preparations, hexamethonium usually attenuated the oral contraction of the LM and CM. However, in the remaining 87% of anal preparations, hexamethonium had no effect on the anal contraction of the LM and CM. 5. Oral and anal reflexes that were hexamethonium resistant were either abolished or attenuated by the further addition of the P2 purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) or alpha,beta-methylene ATP (50-100 microM) to the intermediate chamber. 6. 1,1-Dimethyl-4-phenyl-piperazinium iodide (DMPP, 20 microM) or alpha,beta-methylene ATP (50-100 microM) stimulated both ascending and descending excitatory pathways, when applied to the intermediate chamber. 7. In conclusion, ascending and descending neuro-neuronal transmission in excitatory nervous pathways to the LM and CM is complex and clearly involves neurotransmitter(s) other than acetylcholine (ACh). We suggest mucosal stimulation releases ACh and ATP in both ascending and descending excitatory reflex pathways that synapse with excitatory motoneurons to the LM and CM.

The gastric sling (oblique) muscle (GSM), located close to the lower esophageal sphincter (LES), is involved in gastric motor function and may cooperate with the LES in controlling propulsion between the esophagus and stomach. Neuronal pathways and transmission to the GSM were investigated in isolated esophagus-stomach preparations by using intracellular recording with the focal electrical stimulation and neuroanatomical tracing method. Focal stimulation on the GSM evoked inhibitory junction potentials (IJPs) that were reduced to 45% by 100 microM N-nitro-L-arginine and subsequently blocked by 0.5 microM apamin, thereby unmasking excitatory junction potentials (EJPs), which were abolished by 1 microM hyoscine. Vagal and esophageal stimulation evoked IJPs that were blocked by 100 microM hexamethonium. Vagal stimulation also evoked EJPs after blockade of IJPs. Application of 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate to the GSM labeled muscle motor neurons located in the stomach mainly close to the GSM, with a few neurons (2%) in the esophagus. The majority (79%) of labeled neurons were immunoreactive for choline acetyltransferase and, hence, excitatory motor neurons. Inhibitory motor neurons (nitric oxide synthase immunoreactive; 15%) were clustered in the midline near the gastroesophageal region. These results demonstrate that the GSM is innervated primarily by gastric excitatory and inhibitory motor neurons and some esophageal neurons. Both excitatory (acetylcholine) and inhibitory (nitric oxide and apamin-sensitive component) transmission can be activated via vagal-enteric pathways.

Since activation of cannabinoid CB1 receptors inhibits gastrointestinal transit in the mouse, this study analyzed the action of the cannabinoid receptor agonist methanandamide on distension-induced propulsive motility. Peristalsis in luminally perfused segments of the guinea-pig isolated ileum was elicited by a rise of the intraluminal pressure. The pressure threshold at which peristaltic contractions were triggered was used to quantify drug effects. Methanandamide (0.1-3 microM) inhibited peristalsis as deduced from a concentration-related increase in the peristaltic pressure threshold, an action that was prevented by the CB1 receptor antagonist SR141716A (1 microM) per se, which had no effect on peristalsis. The distension-induced ascending reflex contraction of the circular muscle was likewise depressed by methanandamide in a SR141716A-sensitive manner, whereas indomethacin-induced phasic contractions of the circular muscle were left unchanged by methanandamide. The anti-peristaltic action of methanandamide was inhibited by apamin (0.5 microM), attenuated by N-nitro-L-arginine methyl ester (300 microM) and left unaltered by suramin (300 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (150 microM) and naloxone (0.5 microM). It is concluded that methanandamide depresses intestinal peristalsis via activation of CB1 receptors on enteric neurons, which results in blockade of excitatory motor pathways and facilitation of inhibitory pathways operating via apamin-sensitive K+ channels and nitric oxide.

The effect of muscarinic cholinoceptor antagonists was investigated on the ascending neural pathways activated by electrical stimulation in the guinea-pig ileum. For comparison, prejunctional and postjunctional effects of muscarinic cholinoceptor antagonists were also studied on circular smooth muscle. A two-compartment (oral and anal compartments) bath was used to study the ascending neural pathways. These were activated by electrical field stimulation in the anal compartment and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded isotonically. Pirenzepine (10-300 nM), a muscarinic M1 cholinoceptor antagonist, reduced the ascending neural contractions in a concentration-dependent fashion when applied either to the oral or anal compartments (11-52% and 13-55% inhibition, respectively, P < 0.05). Pirenzepine inhibited (31+/-7%, P < 0.05) the acetylcholine (100 nM)-induced contractions at a higher non-selective concentration (300 nM), while its effect on the electrically-induced contractions was biphasic (10 and 30nM: 8-15% increase, P<0.05; 100 and 300 nM: 16-28% inhibition, P<0.05). The muscarinic M2 cholinoceptor antagonist methoctramine (3-100 nM) did not modify the contractions produced by 100 nM acetylcholine, electrically-induced contractions and the ascending neural contractions (when applied to either compartment). Parafluorohexahydrosiladifenidol (3-100 nM), a muscarinic M3 cholinoceptor antagonist, inhibited the contractions produced by 100 nM acetylcholine (19-81% and 15-69%), electrically-induced contractions (11-71% and 12-72%) and the ascending neural contractions (13-76% and 866%) when applied to the oral compartment, but it was without effect when applied to the anal compartment. These studies suggest that in the enteric ascending neural pathway, muscarinic M1 receptors are involved in neuroneuronal transmission, muscle contraction is mediated by muscarinic M3 cholinergic receptors, whereas muscarinic M2 receptors do not seem to participate.

1. We report the first simultaneous mechanical reflex responses of the longitudinal muscle (LM) and circular muscle (CM) layers of the guinea-pig ileum following mucosal stimulation and distension in vitro. 2. Dissection techniques were used to prevent mechanical interaction between the LM and CM layers both oral and anal to a stimulus site. 3. All graded stimuli produced graded contractions of both the LM and CM orally and anally to the stimulus. Contractions occurred synchronously in the LM and CM and under no circumstances were inhibitory responses recorded in either muscle layer, despite the presence of ongoing cholinergic tone in both the LM and CM. Contractions were abolished by tetrodotoxin (1.6 microM). 4. Local brush stroking of the mucosa evoked a peristaltic wave which readily conducted distally over 13 cm, without the presence of fluid in the lumen. No descending relaxation was observed. 5. Apamin (300 nM) disrupted evoked peristaltic waves and significantly increased the rate-of-rise of the LM and CM contractions anal to a stimulus, and the LM oral to a stimulus. 6. Nomega-nitro-L-arginine (100 microM), a nitric oxide synthesis inhibitor, had no overall significant effect on the characteristics of the LM and CM contractions, although on occasion an enhancement in their peak amplitude was noted. 7. It is suggested that the guinea-pig ileum does not conform to the 'law of the intestine' as postulated by Bayliss & Starling (1899). Rather, local physiological stimulation of the ileum elicits a contraction both orally and anally to a stimulus, which occurs synchronously in both the CM and LM layers. Apamin-sensitive inhibitory neurotransmission modulates the rate-of-rise of the anal contraction of the CM, possibly to generate distal propulsion.

1. Pituitary adenylate cyclase-activating peptide (PACAP) is known to influence the activity of intestinal smooth muscle. This study set out to examine the action of PACAP on normal and drug-inhibited peristalsis and to shed light on its site and mode of action. 2. Peristalsis in isolated segments of the guinea-pig small intestine was elicited by distension through a rise of the intraluminal pressure. Drug-induced motility changes were quantified by alterations of the peristaltic pressure threshold at which aborally moving peristaltic contractions were triggered. 3. PACAP (1-30 nM) stimulated normal peristalsis as deduced from a concentration-related decrease in the peristaltic pressure threshold (maximum decrease by 55%). The peptide's stimulant effect remained intact in segments pre-exposed to apamin (0.5 microM), N-nitro-L-arginine methyl ester (300 microM), naloxone (0.5 microM), atropine (1 microM) plus naloxone (0.5 microM) or hexamethonium (100 microM) plus naloxone (0.5 microM). 4. PACAP (10 nM) restored peristalsis blocked by morphine (10 microM), noradrenaline (1 microM) or N6-cyclopentyladenosine (0.3 microM) and partially reinstated peristalsis blocked by Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine (100 microM) but failed to revive peristalsis blocked by hexamethonium (100 microM) or atropine (1 microM). The peptide's spectrum of properistaltic activity differed from that of naloxone (0.5 microM) and forskolin (0.3 microM). 5. The distension-induced ascending reflex contraction of the circular muscle was facilitated by PACAP (1-30 nM) which itself evoked transient nerve-mediated contractions of intestinal segment preparations. 6. These data show that PACAP stimulates normal peristalsis and counteracts drug-induced peristaltic arrest by a stimulant action on excitatory enteric motor pathways, presumably at the intrinsic sensory neurone level. The action of PACAP seems to involve multiple signalling mechanisms including stimulation of adenylate cyclase.

Intracellular recordings were used to study the contribution of nicotinic and P2X receptors to synaptic transmission to morphologically identified myenteric neurons of guinea-pig ileum. Hexamethonium (100 microM) abolished fast excitatory synaptic potentials (EPSPs) in all orally projecting neurons, but fast EPSPs in anally projecting neurons were resistant to this antagonist. The non-cholinergic fast EPSPs were virtually abolished by suramin (100 microM). This suggests that P2X receptors are important in descending motility reflexes. However, suramin and hexamethonium together did not affect descending inhibitory reflexes when applied to the site of transmission between interneurons in this pathway. These data suggest that P2X receptors are not involved in transmission between descending interneurons, but may be important for transmission to inhibitory motor neurons.

1. Segments of isolated guinea-pig intestine, 12 mm long, were distended slowly by intraluminal fluid infusion or by mechanical stretch as either a tube or flat sheet. In all cases, at a constant threshold length, a sudden, large amplitude contraction of the circular muscle occurred orally, corresponding to the initiation of peristalsis. 2. Circumferential stretch of flat sheet preparations evoked graded contractions of the longitudinal muscle (the 'preparatory phase'), which were maintained during circular muscle contraction. This suggests that the lengthening reported during the emptying phase of peristalsis is due to mechanical interactions. 3. The threshold for peristalsis was lower with more rapid stretches and was also lower in long preparations (25 mm) compared with short preparations (5-10 mm), indicating that ascending excitatory pathways play a significant role in triggering peristalsis. 4. Stretching a preparation beyond the threshold for peristalsis evoked contractions of increasing amplitude; thus peristalsis is graded above its threshold. However, during suprathreshold stretch maintained at a constant length, contractions of the circular muscle quickly declined in amplitude and frequency. 5. Circular muscle cells had a resting membrane potential approximately 6 mV more negative than the threshold for action potentials. During slow circumferential stretch, subthreshold graded excitatory motor input to the circular muscle occurred, prior to the initiation of peristalsis. However, peristalsis was initiated by a discrete large excitatory junction potential (12 +/- 2 mV) which evoked bursts of smooth muscle action potentials and which probably arose from synchronized firing of ascending excitatory neuronal pathways.

The enteric nervous system exerts local control over mixing and propulsive movements in the small intestine. When digestion is in progress, intrinsic primary afferent neurons (IPANs) are activated by the contents of the intestine. The IPANs that have been physiologically characterized are in the intrinsic myenteric ganglia. They are numerous, about 650/mm length of small intestine in the guinea pig, and communicate with each other through slow excitatory transmission to form self-reinforcing assemblies. High proportions of these neurons respond to chemicals in the lumen or to tension in the muscle; physiological stimuli activate assemblies of hundreds or thousands of IPANs. The IPANs make direct connections with muscle motor neurons and with ascending and descending interneurons. The circular muscle contracts as an annulus, about 2-3 mm in minimum oral-to-anal extent in the guinea pig small intestine. The smooth muscle cells form an electrical syncytium that is innervated by about 300 excitatory and 400 inhibitory motor neurons per mm length. The intrinsic nerve circuits that control mixing and propulsion in the small intestine are now known, but it remains to be determined how they are programmed to generate the motility patterns that are observed.

1. The involvement of nitric oxide (NO) in enteric neural pathways underlying reflex responses of the longitudinal muscle (LM) and circular muscle (CM) layers activated by mucosal stimulation was examined in the isolated guinea-pig distal colon. 2. A segment of colon spanned two partitions (10 mm apart), which divided the organ bath into three chambers: a recording chamber where LM and CM tension was measured; a stimulation chamber where mucosal stimulation was applied; and a middle chamber separating them. 3. Brushing the mucosa anal and oral to the recording site evoked simultaneous oral contraction and anal relaxation of both the LM and CM. 4. N omega-nitro-L-argininel-NA; 100 microM) or N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM) applied to the middle chamber or stimulation chamber decreased the oral contractile response of the LM and CM (by about 30-40 %), but increased the anal relaxation (> 600 %) and exposed an anal contraction (> 1000 % increase) of both muscles. The addition of L-NA to the recording chamber reduced the anal relaxation of the LM and CM and the anal contraction of the LM, but slightly increased the anal contraction of the CM. 5. S-Nitroso-N-acetylpenicillamine (SNAP; 10 microM), an NO donor, reversed the effects of L-NA in the middle or stimulation chambers. 6. 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM), a soluble guanylate cyclase inhibitor, mimicked the effects of L-NAin the middle chamber or stimulation chamber, but these effects were not reversed by SNAP. 7. The oral contractile responses, and the anal relaxation and contractile responses of the LM and CM produced by L-NA in the stimulation or middle chambers, were blocked by hexamethonium (300 microM) in any chamber. Atropine (1 microM) in the recording chamber reduced the contractile responses of the LM and CM. 8. In conclusion, endogenous NO facilitates and depresses release of acetylcholine from interneurons in ascending and descending nervous pathways, respectively. These NO effects are mediated through soluble guanylate cyclase in cholinergic interneurons

The lower esophageal sphincter (LES) normally controls the opening and closing of the gastroesophageal junction to resist gastric reflux but allow swallowing. Neuronal pathways controlling the guinea pig LES were investigated anatomically and physiologically in isolated preparations.

Intracellular recording from the LES with focal electrical stimulation and retrograde and anterograde neuronal tracing were used.

Electrical stimulation on the LES evoked inhibitory junction potentials (IJPs), which were reduced by 60% by 100 micromol/L N-nitro-L-arginine and subsequently blocked by 0.5 micromol/L apamin, unmasking excitatory junction potentials, which were abolished by 1 micromol/L hyoscine. Esophageal or vagal stimulation evoked IJPs, which were blocked by 100 micromol/L hexamethonium. Focal stimulation of the upper stomach evoked IJPs at 5-8 of 20 stimulation sites, which were abolished by cutting between the stimulation site and sphincter. Application of 1,1'-didodecyl-3,3,3', 3'-tetramethyl indocarbocyanine perchlorate (DiI) to the gastric sling muscle anterogradely labeled many motor axons in the sling muscle but few in the LES, confirming that the two muscles are separately innervated. DiI on the esophagus labeled nerve fibers, but not cell bodies, in the upper stomach.

The inhibitory motor neurons of the LES receive inputs from the vagus nerve, esophagus, and upper stomach.

1. The role of NK1 and NK3 receptors in synaptic transmission between myenteric neurons during motility reflexes in the guinea-pig ileum was investigated by recording intracellularly the reflex responses of the circular muscle to distension or compression of the mucosal villi. Experiments were performed in a three-chambered organ bath that enabled drugs to be selectively applied to different sites along the reflex pathways. 2. When applied in the recording chamber, an NK1 receptor antagonist, SR140333 (100 nM), reduced by 40-50% the amplitudes of inhibitory junction potentials (i.j.ps) evoked in the circular muscle by activation of descending reflex pathways. This effect was abolished when synaptic transmission in the stimulus region was blocked with physiological saline containing 0.1 mM Ca2+ plus 10 mM Mg2+, leaving only the component of the descending reflex pathway conducted via long anally directed collaterals of intrinsic sensory neurons. 3. SR140333 (100 nM) had no effect on descending reflex i.j.ps when applied to the stimulus region. Ascending reflexes were also unaffected by SR140333 in the stimulus region or between the stimulus and recording sites. 4. Septide (10 nM), an NK1 receptor agonist, enhanced descending reflexes by 30-60% when in the recording chamber. [Sar9,Met(O2)11]substance P had no effect at 10 nM, but potentiated distension-evoked reflexes at 100 nM. 5. A selective NK3 receptor antagonist, SR142801 (100 nM), when applied to the stimulus region, reduced the amplitude of descending reflex responses to compression by 40%, but had no effect on responses to distension. SR142801 (100 nM) had no effect when applied to other regions of the descending reflex pathways. 6. SR142801 (100 nM) only inhibited ascending reflexes when applied at the recording site. However, after nicotinic transmission in the stimulus region was blocked, SR142801 (100 nM) at this site reduced responses to compression. 7. Contractions of the circular muscle of isolated rings of ileum evoked by low concentrations of septide, but not [Sar9,Met(O2)11]substance P, were potentiated by tetrodotoxin (300 nM). 8. Contractile responses evoked by an NK3 receptor agonist, senktide, were non-competitively inhibited by SR142801. After excitatory neuromuscular transmission was blocked, senktide produced inhibitory responses that were also antagonised by SR142801, but to a lesser extent and in an apparently competitive manner. 9. These results indicate that tachykinins acting via NK1 receptors partly mediate transmission to inhibitory motor neurons. NK3 receptors play a role in transmission from intrinsic sensory neurons and from ascending interneurons to excitatory motor neurons during motility reflexes.

The tachykinins substance P and neurokinin A are excitatory cotransmitters of cholinergic enteric neurons, their actions being mediated by NK1, NK2 and NK3 receptors. This study examined which of these receptors are part of the neural circuitry of peristalsis. Peristaltic propulsion in luminally perfused segments of the guinea-pig isolated ileum was elicited by a rise of the intraluminal pressure. The pressure threshold at which peristaltic contractions were triggered was used to quantify drug effects on peristalsis, inhibition of peristalsis being reflected by an increase in the pressure threshold. The NK1, NK2 and NK3 receptor antagonists SR-140333, SR-48968 and SR-142 801 (each at 0.1 microM), respectively, had little effect on peristaltic activity as long as cholinergic transmission was left intact. However, both the NK1 and NK2 receptor antagonist (each at 0.1 microM) abolished peristalsis after cholinergic transmission via muscarinic receptors had been blocked by atropine (1 microM) and peristalsis rescued by naloxone (0.5 microM). When cholinergic transmission via nicotinic receptors was suppressed by hexamethonium (100 microM) and peristalsis restored by naloxone (0.5 microM), only the NK2 receptor antagonist (0.1 microM) was able to attenuate peristaltic performance as deduced from a rise of the peristaltic pressure threshold by 106%. The NK3 receptor antagonist (0.1 microM) lacked a major influence on peristalsis under any experimental condition. It is concluded that tachykinins acting via NK1 and NK2 receptors sustain intestinal peristalsis when cholinergic neuroneuronal and neuromuscular transmission via muscarinic receptors has been suppressed. NK2 receptors help maintaining peristalsis once cholinergic neuroneuronal transmission via nicotinic receptors has been blocked, whereas NK3 receptors play little role in the neural pathways of peristalsis.

1. Orally projecting, cholinergic interneurones are important in mediating ascending excitatory reflexes in the small intestine. We have shown that there is just one major class of orally projecting interneurone, which we have characterized using retrograde labelling in organ culture, combined with immunohistochemistry, intracellular recording and dye filling. 2. Orally projecting interneurones, previously shown to be immunoreactive for choline acetyltransferase, tachykinins, enkephalin, calretinin and neurofilament protein triplet, have axons up to 14 mm long and are the only class of cells with orally directed axons more than 8.5 mm long. 3. They are all small Dogiel type I neurones with short dendrites, usually lamellar in form, and a single axon which sometimes bifurcates. Their axons give rise to short varicose collaterals in myenteric ganglia more than 3 mm oral to their cell bodies. 4. Orally projecting interneurones receive prominent fast excitatory post synaptic potentials (fast EPSPs). A major source of fast EPSPs is other ascending interneurones located further aborally. They also receive fast EPSPs from circumferential pathways. 5. In the stretched preparations used in this study, orally projecting interneurones were highly excitable, firing repeatedly to depolarizing current pulses and had negligible long after-hyperpolarizations following their action potentials. They did not receive measurable non-cholinergic slow excitatory synaptic inputs. 6. Ascending interneurones had a characteristic inflection in their membrane responses to depolarizing current pulses and their first action potential was typically delayed by approximately 30 ms. Under single electrode voltage clamp, ascending interneurones had a transient outward current when depolarized above -70 mV from more hyperpolarized holding potentials. Ascending interneurones also consistently showed marked inward rectification under both current clamp and voltage clamp conditions. 7. This class of cells has consistent morphological, neurochemical and electrophysiological characteristics and are important in mediating orally directed enteric reflexes.

Recent electrophysiological studies of the properties of intestinal reflexes and the neurons that mediate them indicate that the intrinsic sensory neurons may transmit to second order neurons via either fast (30-50 ms duration) or slow (10-60 s duration) excitatory synaptic potentials or both. Which of these possible modes of transmission is involved in the initiation of motility reflexes has not been determined and it is not clear and what the consequences of the different forms of synaptic transmission would be for the properties of the reflex pathways. In the present study, this question has been addressed by the use off a suite of computer programs, Plexus, which was written to simulate the activity of the neurons of the enteric nervous system during intestinal reflexes. The programs construct a simulated enteric nerve circuit based on anatomical and physiological data about the number, functions and interconnections of neurons involved in the control of motility. The membrane potentials of neurons are calculated individually from physiological data about the reversal potentials and membrane conductances for Na+, K+ and Cl-. Synaptic potentials are simulated by changes in specific conductances based on physiological data. The results of each simulation are monitored by recording the membrane potentials of up to 16 separate defined neurons and by recording the summed activity of whole classes of neurons as a function of time and location in the stimulated network. The present series of experiments simulated the behaviour of a network consisting of 18,898 sensory neurons and 3708 ascending interneurons after 75% of the sensory neurons lying in the anal 10 mm of a 30 mm long segment of small intestine were stimulated once. The results were compared with electrophysiological data recorded from myenteric neurons during ascending reflexes evoked either by distension or mechanical stimulation of the mucosa. When transmission from sensory neurons to ascending interneurons was via fast excitatory synaptic potentials, the latencies and durations of the simulated responses were too brief to match the electrophysiologically recorded responses. When transmission from sensory neurons was via slow excitatory synaptic potentials, the latencies were very similar to those recorded physiologically, but the durations of the stimulated responses were much longer than seen in physiological experiments. The latencies and durations of simulated and physiologically recorded responses matched only when the firing of ascending interneurons was limited to the beginning of a slow excitatory synaptic (in this study by limiting the duration of the decrease in K+ conductance). The simulation provided several physiologically testable predictions, indicating that Plexus is an important tool for the investigation of the properties and behaviour of the enteric nervous system.

1. The effect of papaverine, a well known smooth muscle relaxant, was investigated on neural transmission within the enteric nervous system. Segments of guinea-pig ileum were placed in a partitioned bath to enable drugs, including papaverine, to be applied to enteric nerve pathways without interfering with the recording of the smooth muscle contraction. Ascending excitatory enteric nerve pathways were activated by electrical field stimulation in the anal compartment (10 Hz for 2 s, 45 mA, 0.5 ms pulse duration) and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded isotonically. 2. Tetrodotoxin (0.6 microM) and hexamethonium (100 microM) both abolished, or greatly reduced, the contractions when applied to either compartment indicating that nicotinic synapses are involved in this pathway. 3. Papaverine (0.3-30 microM) applied independently to each compartment depressed in a concentration-dependent manner, the nerve-mediated contractions. The IC50 of this inhibitory effect was 3.53 microM for the oral and 4.76 microM for the anal compartments, respectively. Two other phosphodiesterase (PDE) inhibitors, 3-isobutyl-1-methylxanthine (IBMX 10-300 microM) and theophylline (30-1000 microM) added to the anal compartment also inhibited the nerve mediated contractions. Papaverine applied to the anal bath, after IBMX 100 microM (or theophylline 300 microM) further inhibited the nerve-mediated contractions, but was less effective than when applied alone. 4. Phentolamine (1 microM), an alpha-adrenoceptor antagonist, reduced the inhibitory effect of papaverine, but not that of IBMX (100 microM) or theophylline (300 microM). A combination of phentolamine and IBMX (or theophylline) prevented the inhibitory effect of papaverine. 5. Tetrodotoxin, but not papaverine or hexamethonium, inhibited the contraction elicited by electrical stimulation just anal to the partition indicating that papaverine did not affect the generation or conduction of nerve action potentials. 6. Verapamil (1 microM) and nifedipine (1 microM), two smooth muscle relaxants which act by blocking L-type calcium channels, only inhibited the contractions when applied directly to the recording (oral) compartment. This indicates that L-type Ca2+ channels are probably not involved in synaptic transmission in these ascending pathways and thus that the PDE inhibitors do not inhibit synaptic transmission by acting on these channels. omega-Conotoxin GVIA (10 nM), a potent inhibitor of the N-type Ca2+ channels, blocked the nerve-mediated contractions applied to either compartment. Whether the PDE inhibitors exert their inhibitory actions via these channels remains to be established. 7. The results indicate that the PDE inhibitors, papaverine, IBMX and theophylline inhibit excitatory enteric neural pathways by depressing synaptic transmission. The inhibitory effect of papaverine (but not IMBX or theophylline) involves, at least in part, the release of noradrenaline from sympathetic nerves acting on alpha-adrenoceptors on enteric neurones.