Fast synaptic excitatory neurotransmission in the human submucosal plexus.

BACKGROUND: Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. METHODS: We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. KEY RESULTS: Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7 ± 2.4% of all submucosal neurons (n = 6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200 µM) reduced nerve-evoked chloride secretion by 34.3 ± 18.6% (n = 14 patients), whereas D-APV had no effect. CONCLUSION & INFERENCE: Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

Zinc Transporter 3 (ZnT3) in the Enteric Nervous System of the Porcine Ileum in Physiological Conditions and during Experimental Inflammation.

Zinc transporter 3 (ZnT3) is a member of the solute-linked carrier 30 (SLC 30) zinc transporter family. It is closely linked to the nervous system, where it takes part in the transport of zinc ions from the cytoplasm to the synaptic vesicles. ZnT3 has also been observed in the enteric nervous system (ENS), but its reactions in response to pathological factors remain unknown. This study, based on the triple immunofluorescence technique, describes changes in ZnT3-like immunoreactive (ZnT3-LI) enteric neurons in the porcine ileum, caused by chemically-induced inflammation. The inflammatory process led to a clear increase in the percentage of neurons immunoreactive to ZnT3 in all "kinds" of intramural enteric plexuses, i.e., myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses. Moreover, a wide range of other active substances was noted in ZnT3-LI neurons under physiological and pathological conditions, and changes in neurochemical characterisation of ZnT3⁺ cells in response to inflammation depended on the "kind" of enteric plexus. The obtained results show that ZnT3 is present in the ENS in a relatively numerous and diversified neuronal population, not only in physiological conditions, but also during inflammation. The reasons for the observed changes are not clear; they may be connected with the functions of zinc ions and their homeostasis disturbances in pathological processes. On the other hand, they may be due to adaptive and/or neuroprotective processes within the pathologically altered gastrointestinal tract.

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Regenerative Capacity of the Enteric Nervous System after Ileoileal Anastomoses in a Rat Model.

Purpose The aim of the study was to investigate the regeneration and migration of neuronal progenitor cells of the enteric nervous system during wound healing after intestinal anastomosis in the rat ileum. Methods Experiments were performed in a rat model of ileoileal anastomosis. Rats were humanely killed on day 2 or day 10 after anastomosis, and the anastomotic region was compared with ileum of healthy rats. Immunofluorescent staining was performed with protein gene product 9.5, nestin, and S100 antibodies. Ganglia of the anastomotic region in both the myenteric and submucosal plexus were counted, and their diameters were measured and compared between groups. Results Analysis of number and diameter of ganglia in both myenteric and submucosal plexus showed individual alterations as a reaction to the surgical manipulation. Significantly less ganglia were found in the submucosal plexus in the operated groups at both day 2 (p < 0.01) and day 10 (p < 0.01) than in the control group. In the myenteric plexus in the operated group, there was a difference in the number of ganglia at day 2, but ganglia count had recovered at day 10 and was not significantly different from the control group. However, the diameter of ganglia in the myenteric plexus still significantly decreasing on day 10 after surgery than in the control group (p = 0.046). Nestin and S100 double-staining showed an increased expression of nestin around the anastomotic wound. Conclusion Our findings suggest a regenerative potential of the enteric nervous system after the surgical ileoileal anastomosis. The myenteric plexus appears to recover faster than the submucosal plexus. This recovery might be driven by nestin-positive neuronal progenitor cells.

Motor patterns of the small intestine explained by phase-amplitude coupling of two pacemaker activities: the critical importance of propagation velocity.

Phase-amplitude coupling of two pacemaker activities of the small intestine, the omnipresent slow wave activity generated by interstitial cells of Cajal of the myenteric plexus (ICC-MP) and the stimulus-dependent rhythmic transient depolarizations generated by ICC of the deep muscular plexus (ICC-DMP), was recently hypothesized to underlie the orchestration of the segmentation motor pattern. The aim of the present study was to increase our understanding of phase-amplitude coupling through modeling. In particular the importance of propagation velocity of the ICC-DMP component was investigated. The outcome of the modeling was compared with motor patterns recorded from the rat or mouse intestine from which propagation velocities within the different patterns were measured. The results show that the classical segmentation motor pattern occurs when the ICC-DMP component has a low propagation velocity (<0.05 cm/s). When the ICC-DMP component has a propagation velocity in the same order of magnitude as that of the slow wave activity (∼1 cm/s), cluster type propulsive activity occurs which is in fact the dominant propulsive activity of the intestine. Hence, the only difference between the generation of propagating cluster contractions and the Cannon-type segmentation motor pattern is the propagation velocity of the low-frequency component, the rhythmic transient depolarizations originating from the ICC-DMP. Importantly, the proposed mechanism explains why both motor patterns have distinct rhythmic waxing and waning of the amplitude of contractions. The hypothesis is brought forward that the velocity is modulated by neural regulation of gap junction conductance within the ICC-DMP network.

Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X1, P2X2, P2X3 channels, P2Y and A3 metabotropic receptors in neurotransmission.

RATIONALE: The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH: LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS: Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-ß,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPßS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,ß-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS: Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

Properties of cholinergic and non-cholinergic submucosal neurons along the mouse colon.

Submucosal neurons are vital regulators of water and electrolyte secretion and local blood flow in the gut. Due to the availability of transgenic models for enteric neuropathies, the mouse has emerged as the research model of choice, but much is still unknown about the murine submucosal plexus. The progeny of choline acetyltransferase (ChAT)-Cre × ROSA26(YFP) reporter mice, ChAT-Cre;R26R-yellow fluorescent protein (YFP) mice, express YFP in every neuron that has ever expressed ChAT. With the aid of the robust YFP staining in these mice, we correlated the neurochemistry, morphology and electrophysiology of submucosal neurons in distal colon. We also examined whether there are differences in neurochemistry along the colon and in neurally mediated vectorial ion transport between the proximal and distal colon. All YFP(+) submucosal neurons also contained ChAT. Two main neurochemical but not electrophysiological groups of neurons were identified: cholinergic (containing ChAT) or non-cholinergic. The vast majority of neurons in the middle and distal colon were non-cholinergic but contained vasoactive intestinal peptide. In the distal colon, non-cholinergic neurons had one or two axons, whereas the cholinergic neurons examined had only one axon. All submucosal neurons exhibited S-type electrophysiology, shown by the lack of long after-hyperpolarizing potentials following their action potentials and fast excitatory postsynaptic potentials (EPSPs). Fast EPSPs were predominantly nicotinic, and somatic action potentials were mediated by tetrodotoxin-resistant voltage-gated channels. The size of submucosal ganglia decreased but the proportion of cholinergic neurons increased distally along the colon. The distal colon had a significantly larger nicotinic ion transport response than the proximal colon. This work shows that the properties of murine submucosal neurons and their control of epithelial ion transport differ between colonic regions. There are several key differences between the murine submucous plexus and that of other animals, including a lack of conventional intrinsic sensory neurons, which suggests there is an incomplete neuronal circuitry within the murine submucous plexus.

Organization of the innervation of the oesophagus and stomach in chinchilla (Chinchilla laniger, Molina).

Using the histochemical and histological techniques the morphology and topography of the autonomic plexuses of the oesophagus and stomach in chinchilla (Chinchilla laniger) was investigated. AChE-positive and adrenergic structures in the myenteric and submucosal plexus were observed. According to the results the cholinergic structures were formed by the different in shape network meshes, various size of the neurons agglomerations and nerve fibres. Adrenergic fibres were found. Density of these structures varied in different parts of the investigated organs. This study is the first attempt to localize the intramural autonomic plexuses in this species.

Nerve activity recordings in routine human intestinal biopsies.

BACKGROUND: Most direct understanding of enteric nerve (patho)physiology has been obtained by electrode and imaging techniques in animal models and human surgical samples. Until now, neuronal activity recordings from a more accessible human tissue source have remained a true challenge. OBJECTIVES: To record nerve activity in human intestinal biopsies using imaging techniques. DESIGN: Submucous plexus was isolated from duodenal biopsies. Enteric nerves were functionally and morphologically examined using calcium (Ca(2+)) imaging and immunohistochemistry. Exogenous application of high-K(+) solution, the nicotinic cholinergic receptor agonist (1,1-dimethyl-4-phenylpiperazinium; DMPP) or serotonin (5-HT), and electrical stimulation of interganglionic fibre tracts were used to activate the neurons, and intracellular Ca(2+) concentrations ([Ca(2+)](i)) were monitored. Enteric ganglia were stained with neuronal and glial markers. RESULTS: Using high-K(+) solution, 146 neurons were identified in 70 ganglia (44 biopsies from 29 subjects). The exogenous application of DMPP or 5-HT caused a transient [Ca(2+)](i) increase, respectively, in 68% and 63% of the neurons identified by high-K(+). Electrical stimulation evoked responses in 57% of the neurons; these responses were totally or partly suppressed by tetrodotoxin or zero-Ca(2+) solution, respectively. Immunohistochemical analysis showed both isolated neurons and ganglia interconnected by typical interganglionic fibre bundles. The average number of ganglia was 7.7±6.0 per biopsy and each ganglion contained on average 4.5±1.2 neurons. CONCLUSION: In this study, for the first time, live recordings were performed of nerve activity in intestinal biopsies. This novel approach is of key importance to study living neurons in both health and disease and to test newly developed compounds in an in-vitro human tissue model.

Rapid high-amplitude circumferential slow wave propagation during normal gastric pacemaking and dysrhythmias.

BACKGROUND: Gastric slow waves propagate aborally as rings of excitation. Circumferential propagation does not normally occur, except at the pacemaker region. We hypothesized that (i) the unexplained high-velocity, high-amplitude activity associated with the pacemaker region is a consequence of circumferential propagation; (ii) rapid, high-amplitude circumferential propagation emerges during gastric dysrhythmias; (iii) the driving network conductance might switch between interstitial cells of Cajal myenteric plexus (ICC-MP) and circular interstitial cells of Cajal intramuscular (ICC-IM) during circumferential propagation; and (iv) extracellular amplitudes and velocities are correlated. METHODS: An experimental-theoretical study was performed. High-resolution gastric mapping was performed in pigs during normal activation, pacing, and dysrhythmia. Activation profiles, velocities, and amplitudes were quantified. ICC pathways were theoretically evaluated in a bidomain model. Extracellular potentials were modeled as a function of membrane potentials. KEY RESULTS: High-velocity, high-amplitude activation was only recorded in the pacemaker region when circumferential conduction occurred. Circumferential propagation accompanied dysrhythmia in 8/8 experiments was faster than longitudinal propagation (8.9 vs 6.9 mm s(-1) ; P = 0.004) and of higher amplitude (739 vs 528 µV; P = 0.007). Simulations predicted that ICC-MP could be the driving network during longitudinal propagation, whereas during ectopic pacemaking, ICC-IM could outpace and activate ICC-MP in the circumferential axis. Experimental and modeling data demonstrated a linear relationship between velocities and amplitudes (P < 0.001). CONCLUSIONS & INFERENCES: The high-velocity and high-amplitude profile of the normal pacemaker region is due to localized circumferential propagation. Rapid circumferential propagation also emerges during a range of gastric dysrhythmias, elevating extracellular amplitudes and organizing transverse wavefronts. One possible explanation for these findings is bidirectional coupling between ICC-MP and circular ICC-IM networks.

Ca(2+) transients in submucous neurons during the colonic migrating motor complex in the isolated murine large intestine.

BACKGROUND: The colonic migrating motor complex (CMMC) is a spontaneous, rhythmic, and neurally mediated motor pattern generated by myenteric neurons, which can propel fecal pellets in mice. Our aim was to determine whether submucous neurons were also activated during the CMMC. METHODS: :The isolated murine colon was opened and sections of mucosa were removed to expose the submucous ganglia, which were then loaded with Fluo-4. KEY RESULTS: Colonic migrating motor complexes, which occurred spontaneously or by mechanically stimulating the mucosa, were identified by displacement of the tissue (duration = 23.3 s). Between CMMCs, spontaneous Ca(2+) transients (frequency = 0.9 Hz) were observed in 55% (n = 8) of submucous neurons. During the CMMC, 98% (seven ganglia, n = 7) of submucous neurons within the same ganglion exhibited rapid Ca(2+) transients (1.6 Hz) superimposed on a sustained rise in Ca(2+) (duration ∼23 s) that occurred 1.7 s following the mucosal stimulus; whereas other neurons exhibited a similar, but delayed response that occurred either at 7 or 13 s following the stimulus. The activity in submucous neurons was correlated with activity in adjacent nerve varicosities. Ondansetron (1 mm; 5-HT(3) antagonist) significantly reduced the frequency and duration of the Ca(2+) transient responses. CONCLUSIONS & INFERENCES: Activity in the submucous neurons appears to be secondary to that in the myenteric plexus and appears to be generated largely by activity in myenteric descending (serotonergic) interneurons. During the CMMC, there is likely to be an increase in secretion to lubricate and facilitate fecal pellet propulsion.

Maxi-channels recorded in situ from ICC and pericytes associated with the mouse myenteric plexus.

Ion channels are fundamental to gastrointestinal pacemaking by interstitial cells of Cajal (ICC). Previously, we have recorded a high-conductance chloride channel (HCCC) from ICC, both in culture and in situ, associated with the myenteric plexus. The biophysical properties of the HCCC (conductance, subconductances, voltage- and time-dependent inactivation) suggest it is a member of a class called the maxi-anion channels. In this study we further investigated the properties of the HCCC in situ. Our main finding was that the HCCC is not strictly a chloride channel but has a relative sodium-chloride permeability (P(Na/Cl)) of 0.76 to 1.64 (depending on the method of measurement). Therefore, we have renamed the HCCC the "maxi-channel." A maxi-channel was also expressed by pericytes associated with the vasculature near the myenteric plexus. This had a lower P(Na/Cl) (0.33 to 0.49, depending on the method of measurement) but similar conductance (326 ± 7 vs. 316 ± 24 pS for ICC). This is the first report of cation permeability equaling anion permeability in a maxi-anion channel. As such, the properties of the maxi-channels described in this article may have implications for the maxi-anion channel field, as well as for studies of their role in ICC and pericytes.

Mechanisms underlying distension-evoked peristalsis in guinea pig distal colon: is there a role for enterochromaffin cells?

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.

Leptin excites enteric neurons of guinea-pig submucous and myenteric plexus.

BACKGROUND: Leptin, one of the most prominent mediators released from adipocytes, influences neuronal activity in the central nervous system. The enteric nervous system (ENS) expresses leptin receptors but consequence of activation of these receptors on enteric neuron activity has not been systematically studied. An adipocyte-ENS axis is suggested by close apposition between enteric nerves and adipocytes. The aim of this study was to investigate the effects of leptin on guinea-pig submucous and myenteric neurons. METHODS: Using voltage sensitive dye imaging, we recorded neural responses to application of leptin (0.0625 nmol L(-1)) in myenteric and submucous neurons, nicotine (10 µmol L(-1)) served as a reference for neuronal excitation. Mucosal ion secretion and muscle activity were measured in vitro with Ussing and organ bath techniques, respectively. KEY RESULTS: Leptin induced spike discharge in 13.6% of submucous neurons and in 8.2% of myenteric neurons (1.1 ± 0.9 and 1.2 ± 1.0 Hz, respectively). Although there was an overlap of nicotine and leptin responses, 38.5% of submucous and 25% of myenteric neurons activated by leptin did not respond to nicotine. Leptin did not inhibit ongoing spike discharge or fast excitatory postsynaptic potentials. Leptin (0.0625 nmol L(-1)) did not affect mucosal secretion or muscle activity suggesting a subtle modulatory action of leptin at the level of the ENS. CONCLUSIONS & INFERENCES: Leptin activates submucous and myenteric neurons indicating relevance for adipocyte-ENS signaling. These results set the basis for further studies to reveal the functional correlate of the neural action of leptin in the ENS.

The aging colon: the role of enteric neurodegeneration in constipation.

Constipation is a common problem in the elderly, and abnormalities in the neural innervation of the colon play a significant role in abnormalities in colonic motility leading to delayed colonic transit. The scope of this review encompasses the latest advances to enhance our understanding of the aging colon with emphasis on enteric neurodegeneration, considered a likely cause for the development of constipation in the aging gut in animal models. Neural innervation of the colon and the effects of aging on intrinsic and extrinsic nerves innervating the colonic smooth muscle is discussed. Evidence supporting the concept that neurologic disorders, such as Parkinson's disease, not only affect the brain but also cause neurodegeneration within the enteric nervous system leading to colonic dysmotility is presented. Further research is needed to investigate the influence of aging on the gastrointestinal tract and to develop novel approaches to therapy directed at protecting the enteric nervous system from neurodegeneration.

5-HT(1A), SST(1), and SST(2) receptors mediate inhibitory postsynaptic potentials in the submucous plexus of the guinea pig ileum.

Vasoactive intestinal peptide (VIP) immunoreactive neurons are important secretomotor neurons in the submucous plexus. They are the only submucosal neurons to receive inhibitory inputs and exhibit both noradrenergic and nonadrenergic inhibitory synaptic potentials (IPSPs). The former are mediated by alpha(2)-adrenoceptors, but the receptors mediating the latter have not been identified. We used standard intracellular recording, RT-PCR, and confocal microscopy to test whether 5-HT(1A), SST(1), and/or SST(2) receptors mediate nonadrenergic IPSPs in VIP submucosal neurons in guinea pig ileum in vitro. The specific 5-HT(1A) receptor antagonist WAY 100135 (1 microM) reduced the amplitude of IPSPs, an effect that persisted in the presence of the alpha(2)-adrenoceptor antagonist idazoxan (2 microM), suggesting that 5-HT might mediate a component of the IPSPs. Confocal microscopy revealed that there were many 5-HT-immunoreactive varicosities in close contact with VIP neurons. The specific SSTR(2) antagonist CYN 154806 (100 nM) and a specific SSTR(1) antagonist SRA 880 (3 microM) each reduced the amplitude of nonadrenergic IPSPs and hyperpolarizations evoked by somatostatin. In contrast with the other antagonists, CYN 154806 also reduced the durations of nonadrenergic IPSPs. Effects of WAY 100135 and CYN 154806 were additive. RT-PCR revealed gene transcripts for 5-HT(1A), SST(1), and SST(2) receptors in stripped submucous plexus preparations consistent with the pharmacological data. Although the involvement of other neurotransmitters or receptors cannot be excluded, we conclude that 5-HT(1A), SST(1), and SST(2) receptors mediate nonadrenergic IPSPs in the noncholinergic (VIP) secretomotor neurons. This study thus provides the tools to identify functions of enteric neural pathways that inhibit secretomotor reflexes.

Release of 5-hydroxytryptamine from the mucosa is not required for the generation or propagation of colonic migrating motor complexes.

BACKGROUND & AIMS: The pacemaker mechanism that underlies the cyclic generation of colonic migrating motor complexes (CMMCs) is unknown, although studies have suggested that release of 5-hydroxytryptamine (5-HT) from enterochromaffin cells in the mucosa is essential. However, no recordings of 5-HT release from the colon have been made to support these suggestions. METHODS: We used real-time amperometry to record 5-HT release directly from the mucosa in mouse isolated colon to determine whether 5-HT release from enterochromaffin cells was required for CMMC generation. RESULTS: We found that 5-HT was released from mucosal enterochromaffin cells during many, but not all, CMMC contractions. However, spontaneous CMMCs still were recorded even after removal of the mucosa, and submucosa and submucosal plexus when all release of 5-HT had been abolished. CMMC pacemaker frequency was slower in the absence of the mucosa, an effect reversed by focal application of exogenous 5-HT onto the myenteric plexus. Despite the absence of the mucosa and all detectable release of 5-HT, ondansetron significantly reduced CMMC frequency, suggesting that 5-HT(3) receptor blockade slows the CMMC pacemaker via a mechanism independent of 5-HT release from enterochromaffin cells. CONCLUSIONS: Our results show that 5-HT can be released dynamically during CMMCs. However, the intrinsic pacemaker and pattern generator underlying CMMC generation lies within the myenteric plexus and/or muscularis externa and does not require any release of 5-HT from enterochromaffin cells. Endogenous release of 5-HT from enterochromaffin cells plays a modulatory role, not an essential role, in CMMC generation.

Cholera toxin induces sustained hyperexcitability in submucosal secretomotor neurons in guinea pig jejunum.

BACKGROUND & AIMS: Neural mechanisms underlying cholera toxin (CT)-induced intestinal hypersecretion remain unclear. We investigated long-term excitability changes in vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) secretomotor neurons after prolonged luminal exposure to CT. METHODS: Isolated segments of guinea pig jejunum were incubated with saline or CT +/- neurotransmitter antagonist in the lumen; the submucosal plexus was then dissected clear, circumferentially adjacent to intact mucosa. Synaptic inputs and firing properties of S neurons in ganglia next to the mucosa in control saline were studied using intracellular recording. Neurons were processed for VIP and NPY immunoreactivity. RESULTS: Thirty S neurons (20 VIP(+), 7 NPY(+), 3 VIP(-)/NPY(-)) from CT-treated preparations and 27 control S neurons (19 VIP(+), 4 NPY(+), 4 VIP(-)/NPY(-)) in ganglia adjacent to intact mucosa were analyzed. VIP(+) and NPY(+) neurons in CT-treated preparations fired significantly more action potentials and for longer periods during injected depolarizing current pulses (50-350 pA) than control neurons. Addition of tetrodotoxin, hexamethonium, granisetron, or the neurokinin-1 (NK1) antagonist SR140333 during the CT incubation blocked CT-induced effects in both neuron types. The NK3 antagonist SR142801 blocked CT-induced effects in NPY(+) neurons and reduced the number of action potentials in VIP(+) neurons. Synaptic activity was unaffected by CT. CONCLUSIONS: CT induces specific and sustained hyperexcitability of secretomotor neurons in enteric pathways. CT acts in the mucosa. Its effect is neurally mediated and depends on 5-hydroxytryptamine-3, nicotinic, and NK1 receptors. This system represents a unique model to understand the neural mechanisms of action of CT and to identify therapeutic targets.

Synaptic transmission from the submucosal plexus to the myenteric plexus in Guinea-pig ileum.

Stimulation of the myenteric plexus results in activation of submucosal neurons and dilation of arterioles, one way that motility and secretion can be coupled together. The present study aimed to examine the converse, whether myenteric neurons receive synaptic input from the submucosal plexus (SMP). Intracellular recordings were made from guinea-pig ileal myenteric neurons while the SMP was electrically stimulated. Of the 29 neurons studied (13 S and 16 AH neurons), stimulation of the SMP evoked a synaptic potential in only seven cells, or 24% of neurons. When the SMP was situated oral to the myenteric plexus, 4 of 13 (31%) myenteric neurons had synaptic input. When it was situated circumferential, 2 of 8 (25%) had input, and when the SMP was situated anal 1 of 8 (13%) had input. Overall, 5 of the 13 (38%) S neurons responded with fast excitatory post-synaptic potentials (EPSPs), one of which also showed a slow EPSP, while 2 of the 16 (13%) AH neurons responded with a slow EPSP. This study indicates that the synaptic input from the SMP to myenteric neurons is relatively sparse. Whether this input is less important than the myenteric to submucosal input or simply represents a more selective form of control is unknown.