The relationship between inflammation‐induced neuronal excitability and disrupted motor activity in the guinea pig distal colon

Background Colitis is associated with increased excitability of afterhyperpolarization neurons (AH neurons) and facilitated synaptic transmission in the myenteric plexus. These changes are accompanied by disrupted propulsive motility, particularly in ulcerated regions. This study examined the relationship between myenteric AH neuronal hyperexcitability and disrupted propulsive motility. Methods The voltage‐activated Na⁺ channel opener veratridine, the intermediate conductance Ca²⁺‐activated K⁺ channel inhibitor TRAM‐34 and the 5‐HT₄ receptor agonist tegaserod were used to evaluate the effects of neuronal hyperexcitability and synaptic facilitation on propulsive motility in normal guinea pig distal colon. Because trinitrobenzene sulfonic acid (TNBS)‐colitis‐induced hyperexcitability of myenteric afferent neurons involves increases in hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channel activity, the HCN channel inhibitors Cs⁺ and ZD7288 were used to suppress AH neuronal activity in TNBS‐colitis. Key Results In non‐inflamed preparations, veratridine halted propulsive motility (P < 0.001). The rate of propulsive motor activity was significantly reduced following addition of TRAM‐34 and tegaserod (P < 0.001). In TNBS‐inflamed preparations, in which motility was temporarily halted or obstructed at sites of ulceration, both Cs⁺ and ZD7288 normalized motility through the inflamed regions. Immunohistochemistry studies demonstrated that the proportion of AH neurons in the myenteric plexus was unchanged in ulcerated regions, but there was a 10% reduction in total number of neurons per ganglion. Conclusions and Inferences These findings support the concept that inflammation‐induced neuroplasticity in myenteric neurons, involving changes in ion channel activity that lead to enhanced AH neuronal excitability, can contribute to impaired propulsive colonic motility.     

Neuromuscular changes in a rat model of colitis

Intracolonic administration of Trichinella spiralis larvae in rats causes colitis with features similar to ulcerative colitis, notably with inflammation predominantly limited to the colonic mucosa. Our aim was to characterize the functional and neurochemical changes occurring within the myenteric (MP) and submucosal plexuses (SMP) during T. spiralis-induced colitis. Infected rats had decreased body weight, altered stool consistency and elevated myeloperoxidase activity, 6 and 14 days post-infection (PI). Responses to acetylcholine and KCl in circular muscle strips were reduced in infected tissues, demonstrating an impairment of contractility. In addition, there was a decrease in spontaneous motor activity and reduced sensitivity to the nitric oxide synthase (NOS) inhibitor L-NOArg, corresponding with a significant reduction in NOS immunoreactive neurons in the MP of infected animals. T. spiralis did not alter the total number of myenteric or submucosal neurons. Substance P innervation of submucosal blood vessels was reduced after infection, as were submucosal calretinin and calbindin immunoreactive neurons. No changes in choline acetyltransferase and calcitonin gene-related peptide immunoreactivity were observed. T. spiralis-induced colitis causes profound neuromuscular adaptations. The reduction in NOS neurons appears to underlie changes in motility.     

N‐Methyl‐d‐aspartate receptor antagonism decreases motility and inflammatory activation in the early phase of acute experimental colitis in the rat

Background Inflammatory bowel diseases are accompanied by severe motility disorders. The aim of our study was to investigate whether the blockade of peripheral N‐methyl‐D‐aspartate (NMDA)‐sensitive glutamate receptors (NMDA‐Rs) alters motility changes in chemically induced acute colitis and how this modulation is accomplished. Methods The inflammatory and motility changes in 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced colitis were studied in anaesthetized Wistar rats following treatment with the natural NMDA‐R antagonist kynurenic acid (KynA) or SZR‐72, a blood‐brain barrier‐permeable synthetic KynA analogue. The macrohaemodynamics, serosal microcirculation (visualized by intravital videomicroscopy), plasma levels of tumour necrosis factor alpha (TNF‐α), inflammatory enzyme activities (xanthine oxidoreductase (XOR), myeloperoxidase (MPO) and nitric oxide synthase (NOS)), and colonic motility (with a strain‐gauge technique) were evaluated 17 h after colitis induction and compared with the control conditions. Key Results The TNBS enema induced a systemic hyperdynamic circulatory reaction, increased the serosal capillary blood flow, significantly elevated the mucosal XOR, MPO and NOS activities and augmented the colonic motility relative to the controls. The NMDA‐R antagonist treatment with KynA or SZR‐72 significantly reduced the XOR, NOS and MPO activities, decreased the motility and increased the tone of the colon. Conclusions & Inferences These data demonstrate a potential modulatory mechanism of NMDA‐R in altered colonic motility in TNBS colitis. Inhibition of the enteric NMDA‐Rs may provide a therapeutic option via which to influence intestinal hypermotility, microcirculatory changes and inflammatory activation simultaneously.     

Neural mechanisms of early postinflammatory dysmotility in rat small intestine

Although human postinflammatory dysmotility is known, so far animal studies have primarily investigated changes during inflammation. Here, we focused on postinflammatory changes in rat jejunal myenteric plexus and jejunal motility. Evolution of ethanol/2,4,6-tri-nitrobenzene sulphonic acid (TNBS)-induced inflammation was assessed histologically and by measuring myeloperoxidase activity (MPO). Electromyography and immunohistochemistry were performed 1 week after ethanol/TNBS and also after N(G)-nitro-L-arginine methyl ester (L-NAME) administration. Ethanol/TNBS induced a transient inflammation, with normalization of MPO and histological signs of an early phase of recovery after 1 week. The number of cholinergic neurones was not altered, but myenteric neuronal nitric oxide synthase (nNOS)-immunoreactivity was significantly lower in the early phase of recovery after TNBS compared with water (1.8 +/- 0.2 vs 3.5 +/- 0.2 neurones ganglion(-1), P < 0.001). Interdigestive motility was disrupted with a loss of phase 1 quiescence, an increase of migrating myoelectric complex cycle length, a higher number of non-propagated activity fronts and a decrease of adequately propagated phase 3 s after TNBS. Administration of L-NAME resulted in a similar disruption of interdigestive motility patterns. In the early phase of recovery after ethanol/TNBS-induced jejunal inflammation, a loss of motor inhibition occurs due to a decrease of myenteric nNOS activity. These observations may provide a model for early postinflammatory dysmotility syndromes.     

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

Abstract 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.     

Colonic inflammation up-regulates voltage-gated sodium channels in bladder sensory neurons via activation of peripheral transient potential vanilloid 1 receptors

Background Primary sensory neurons express several types of ion channels including transient receptor potential vanilloid 1 (TRPV1) and voltage‐gated Na⁺ channels. Our previous studies showed an increased excitability of bladder primary sensory and spinal neurons triggered by inflammation in the distal colon as a result of pelvic organ cross‐sensitization. The goal of this work was to determine the effects of TRPV1 receptor activation by potent agonists and/or colonic inflammation on voltage‐gated Na⁺ channels expressed in bladder sensory neurons. Methods Sprague–Dawley rats were treated with intracolonic saline (control), resiniferatoxin (RTX, 10^?7 mol L^?1), TNBS (colonic irritant) or double treatment (RTX followed by TNBS). Key Results TNBS‐induced colitis increased the amplitude of total Na⁺ current by two‐fold and of tetrodotoxin resistant (TTX‐R) Na⁺ current by 78% (P ≤ 0.05 to control) in lumbosacral bladder neurons during acute phase (3 days post‐TNBS). Instillation of RTX in the distal colon caused an enhancement in the amplitude of total Na⁺ current at ?20 mV from ?112.1 ± 18.7 pA/pF (control) to ?183.6 ± 27.8 pA/pF (3 days post‐RTX, P ≤ 0.05) without changes in TTX resistant component. The amplitude of net Na⁺ current was also increased by 119% at day 3 in the group with double treatment (RTX followed by TNBS, P ≤ 0.05 to control) which was significantly higher than in either group with a single treatment. Conclusions & Inferences These results provide evidence that colonic inflammation activates TRPV1 receptors at the peripheral sensory terminals leading to an up‐regulation of voltage gated Na⁺ channels on the cell soma of bladder sensory neurons. This mechanism may underlie the occurrence of peripheral cross‐sensitization in the pelvis and functional chronic pelvic pain.     

Myenteric ganglia from the adult guinea-pig small intestine in tissue culture

Abstract Myenteric ganglia dissociated from the small intestine of adult guinea-pigs survived in long-term culture (1–2 months) and progressed to structural organization resembling the myenteric plexus in situ. Developmental changes were similar to cultures derived from neonatal intestine. After one week, the neurons gathered into clusters on a glial cell carpet. Processes from the neurons branched and ramified over the glial substrate. As the cultures matured, the processes joined into tracts and the neurons and glia formed compact aggregates reminiscent of ganglia interconnected by fibre bundles. Injection of dye revealed characteristic Dogiel I and II neuronal morphology. Electrical recording identified electrical and synaptic behaviour comparable to intact myenteric plexus, longitudinal muscle preparations, except slow synaptic excitation was absent. Pharmacological responses to forskolin and 5-hydroxytryptamine were essentially the same as in freshly dissected preparations. Lucifer yellow injected into single glial cells spread to a broad population indicative of the dye coupling found among glia in the myenteric plexus in situ. The results suggest that adult myenteric ganglia in culture are a useful model for investigation of aspects of enteric neurobiology including: (a) formation of connections in microcircuits; (b) cellular neurophysiology of enteric neurons; (c) neuropharmacology; and (4) cell biology of neuronal-glial interactions in the myenteric plexus.     

Prolonged sympathetic innervation of sensory neurons in rat thoracolumbar dorsal root ganglia during chronic colitis

Background Peripheral irritation‐induced sensory plasticity may involve catecholaminergic innervation of sensory neurons in the dorsal root ganglia (DRG). Methods Catecholaminergic fiber outgrowth in the thoracolumbar DRG (T13‐L2) was examined by tyrosine hydroxylase (TH) immunostaining, or by sucrose‐potassium phosphate‐glyoxylic acid histofluorescence method. TH level was examined by Western blot. Colonic afferent neurons were labeled by retrograde neuronal tracing. Colitis was induced by intracolonic instillation of tri‐nitrobenzene sulfonic acid (TNBS). Key Results The catecholaminergic fibers formed ‘basket‐like’ structures around the DRG cells. At 7 days following TNBS treatment, the number of DRG neurons surrounded by TH‐immunoreactive fibers and the protein levels of TH were significantly increased in T13, L1, and L2 DRGs (two‐ to threefold, P < 0.05). The DRG neurons that were surrounded by TH immunoreactivity were 200 kDa neurofilament‐positive, but not isolectin IB4‐positve or calcitonin gene‐related peptide‐positive. The TH‐immunoreactive fibers did not surround but adjoin the specifically labeled colonic afferent neurons, and was co‐localized with glial marker S‐100. Comparison of the level of TH and the severity of colonic inflammation showed that following TNBS treatment, the degree of colonic inflammation was most severe at day 3, subsided at day 7, and significantly recovered by day 21. However, the levels of TH in T13‐L2 DRGs were increased at both 3 days and 7 days post TNBS treatment and persisted up to 21 days (two‐ to fivefold increase, P < 0.05) as examined. Conclusions & Inferences Colonic inflammation induced prolonged catecholaminergic innervation of sensory neurons, which may have relevance to colitis‐induced chronic visceral hypersensitivity and/or referred pain.     

Enteric neuropathy evoked by repeated cisplatin in the rat

Background Acute administration of the antitumoral drug cisplatin can induce nausea/emesis and diarrhea. The long‐term effects of cisplatin on gastrointestinal motility, particularly after repeated administration, are not well known. Because cisplatin is highly neurotoxic, myenteric neurons can be affected. Our aim was to study the prolonged effects of repeated cisplatin administration in a rat model, focusing on gastrointestinal motor function and myenteric neurons. Methods Rats received saline or cisplatin (1 or 3 mg kg^?1, i.p.) once weekly for 5 weeks. One week after treatment, both upper gastrointestinal transit and colonic activity were evaluated, and tissue samples from ileum, colon and rectum were processed for histological analysis. Intestinal transit was measured invasively (charcoal method). Colonic activity was determined electromyographically. The gut wall structure was evaluated in sections using conventional histology and immunohistochemistry. Whole‐mount preparations from the distal colon were labeled for different markers, including nitric oxide synthase (NOS) and calcitonin‐gene related peptide (CGRP) to determine relative proportions of myenteric neurons vs the total neuronal population labeled with HuC/D. Key Results One week after repeated cisplatin exposure, the upper gastrointestinal transit rate and colonic activity were dose‐dependently reduced. The number of NSE‐ or HuC/D‐immunoreactive myenteric neurons per ganglion was decreased; the proportion of CGRP‐immunoreactive neurons was decreased, whereas that of NOS‐immunoreactive cells was increased. Conclusions & Inferences Chronic cisplatin may induce an enteric neuropathy characterized by changes in myenteric neurons associated with marked gastrointestinal motor dysfunction.     

The effects of glucagon‐like peptide 2 on enteric neurons in intestinal inflammation

Background Intestinal inflammation alters the structure and function of the enteric nervous system (ENS). Glucagon‐like peptide 2 (GLP‐2) reduces intestinal inflammation and has trophic effects on isolated neurons. This study examined the effects of GLP‐2 treatment on the submucosal plexus of rat colon in the trinitrobenzene sulfonic acid (TNBS) model of colitis. Methods After administration of TNBS or saline/ethanol for controls, animals were allocated to treatment with GLP‐2 (50 μg kg^?1day^?1, s.c.) or sham injection of vehicle, twice daily. Animals were monitored, following clinical parameters, and killed on day 5. The number of neuronal cell bodies per ganglion was quantified using immunohistochemistry on submucosal whole mount preparations, with further characterization of specific subpopulations using antibodies against vasoactive intestinal polypeptide (VIP), neuronal nitric oxide synthase (nNOS), and enteric glial cells with glial fibrillary acid protein and S100. Key Results Glucagon‐like peptide 2 treatment was associated with a significant amelioration of weight loss, and reduced neutrophil infiltration and microscopic colitis scores in the TNBS animals. Inflammation resulted in a loss of enteric neurons in submucosal ganglia; GLP‐2 treatment restored the enteric neuronal populations to normal. In control, non‐inflamed animals, GLP‐2 treatment increased the number of VIP expressing neurons per ganglion; in TNBS‐treated animals, GLP‐2 prevented an inflammation‐induced reduction in the numbers of VIP expressing neurons per ganglion. Glucagon‐like peptide 2 did not change the numbers of nNOS neurons or enteric glial cells in either the control, or inflamed state. Conclusions & Inferences These findings show that GLP‐2 increased the number of VIP expressing neurons in normal animals, and prevents the inflammation‐induced loss of neurons in the colonic submucosal ganglia, with an increase in the proportion of VIP expressing neurons. They suggest that GLP‐2 may have a role in protecting or regulating the circuitry of the ENS under basal and inflamed states.     

Altered myoelectrical activity in noninflamed ileum of rats with colitis induced by trinitrobenzene sulphonic acid

Changes in gastric emptying and orocaecal transit time in patients with ulcerative colitis suggest that disturbances in gut motility may not be restricted to inflamed sites. This study sought to characterize changes in the motility of noninflamed ileum in a rat colitis model and to explore the mechanism(s) potentially involved. The myoelectrical activity of the ileum was recorded in rats with trinitrobenzene sulphonic acid (TNBS)-induced colitis. The degree of ileal and colonic inflammation was assessed by quantification of macroscopic damage and myeloperoxidase activity (MPO). The effect on ileal motility of pretreatment with atropine, indomethacin and NG-nitro-L-arginine-methyl ester (L-NAME) was investigated. TNBS-induced inflammation was restricted to the distal colon, as evidenced by morphological scores and MPO. Colitis was associated with increased frequency of ileal migrating motor complexes, characterized mainly by a decrease in the duration of phases I and III. The occurrence of ileal giant migrating complexes remained unchanged. The myoelectrical changes observed in the ileum persisted after treatment with atropine, indomethacin and L-NAME. Distal colitis is associated with abnormal myoelectrical activity in the noninflamed ileum of rats. Neither acetylcholine nor prostaglandins and nitric oxide seem to be involved.     

Direct synaptic contacts on the myenteric ganglia of the rat stomach from the dorsal motor nucleus of the vagus

The myenteric ganglia regulate not only gastric motility but also secretion, because a submucous plexus is sparsely developed in the rodent stomach. We have examined whether the neurons of the dorsal motor nucleus of the vagus (DMV) have direct synaptic contacts on the myenteric ganglia and the ultrastructure of the vagal efferent terminals by using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The myenteric ganglia of the rat were composed of four types of neurons, i.e., small, medium-sized, large, and elongated neurons. The average numbers of axosomatic terminals per profile were 2.0 on the small neurons, 3.1 on the medium-sized neurons, 1.2 on the large neurons, and 4.2 on the elongated neuron. More than half of the axosomatic terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized, and large neurons. About 80% of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. When WGA-HRP was injected into the DMV, many anterogradely labeled terminals were found around the myenteric neurons. The labeled terminals were large (3.16 +/- 0.10 microm) and contacted exclusively the somata. Most of them (about 90%) contained round vesicles and formed asymmetric synaptic contacts. Serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the DMV. The vagal axon terminals generally contacted the medium-sized or the elongated neurons, whereas a few labeled terminals contacted the small and the large neurons. The present results indicate that the DMV projects to all types of neurons and that their axon terminals contain mostly round synaptic vesicles and form asymmetric synaptic contacts.     

Ramifications of the axons of AH-neurons injected with the intracellular marker biocytin in the myenteric plexus of the guinea pig small intestine.

The projections and terminal ramifications of electrophysiologically characterized myenteric neurons of the guinea pig small intestine were studied after intracellular injection of the marker substance biocytin. Myenteric neurons were impaled with microelectrodes containing 4% biocytin in 2 M KCl (pH 7.4) and characterized electrophysiologically as either AH-neurons or S-neurons. AH-neurons were neurons in which action potentials were followed by prolonged after-hyperpolarizations (lasting greater than 4 seconds). S-neurons were neurons in which such hyperpolarizations were not seen. Electrical stimulation of internodal strands evoked prominent fast excitatory synaptic potentials in S-neurons, but not in AH-neurons. Biocytin was injected electrophoretically into the impaled AH-neurons by passage of hyperpolarizing current (0.6-0.8 nA for 5-15 minutes) through the recording electrode. The preparation was then fixed in Zamboni's fixative, dehydrated, and exposed to avidin coupled to horseradish peroxidase which allowed the injected biocytin to be visualised via a diaminobenzidine reaction. In many cases, the injected biocytin appeared to fill all the processes of injected AH-neurons that ramified within the myenteric plexus. The filled processes included axons running up to 4 mm within the plexus and profuse varicose terminals ramifying within both the ganglion containing the injected cell body and nearby ganglia. Most (90%) cell bodies of the injected AH-neurons had the morphology of Dogiel type II neurons; large, mostly smooth cell bodies with few short processes and several long processes. The other 10% of the AH-neurons had similar cell bodies and long processes but also had prominent short filamentous processes. This population was termed dendritic AH-neurons. The projections and terminals of 28 AH/Dogiel type II neurons and 7 dendritic AH-neurons were analysed in detail. Both types of neurons project circumferentially to provide terminals to nearby ganglia, but the AH/Dogiel type II neurons also provide terminals to their own ganglia while the dendritic AH-neurons typically do not. Although many of the injected AH-neurons had projections orally or anally along the intestine no evidence for a preferential direction of projection was obtained. Analysis of the areas and distributions of the terminal fields of the AH/Dogiel type II neurons suggests that each may contact several other myenteric neurons and that each myenteric neuron may receive input from about ten AH/Dogiel type II neurons.     

Distribution pattern of tachykinin NK2 receptors in human colon: involvement in the regulation of intestinal motility

Although a number of pharmacological studies have shown the involvement of tachykinin type 2 receptors (NK2r) in the regulation of human colonic motility, few data are available so far on their pattern of expression. In this study this pattern was investigated in the myenteric plexuses, the longitudinal and circular muscle layers (external muscular layers), and the interstitial cells of Cajal (ICCs) using confocal microscopy immunofluorescence methods. NK2r immunoreactivity (NK2r-IR) was detected in the soma of myenteric neurons and in nerve varicosities located in myenteric plexuses as well as in external muscular layers. Colocalization analysis of NK2r-IR and synaptophysin-IR, showed significant regional differences in the distribution of NK2r-expressing nerve varicosities, the rate of occurrence was found to be 56.08% +/- 3% (mean +/- SE) in the external muscular layers and 30.22% +/- 1% (mean +/- SE) in the myenteric plexuses. NK2r-IR was found in membranes of most muscle cells previously incubated with a selective NK2r agonist, [beta-Ala(8)] neurokinin A fragment 4-10, at 4 degrees C, and then mainly relocated in the cytoplasm when heated to 37 degrees C. A number of NK2r-IR nerve varicosities were close to NK2r-expressing neurons and muscle cells. Some of NK2r-expressing neurons and nerves were tachykinin-IR. No NK2r-IR was detected in ICCs. The present data indicate that presynaptic and postsynaptic neuroneuronal and neuromuscular regulatory processes mediated by tachykinins via NK2r may occur for modulating human colonic motility.     

Myenteric plexus injury and apoptosis in experimental colitis

Intestinal inflammatory conditions are associated with structural and functional alterations of the enteric nervous system (ENS). While injury to the enteric nervous system is well described, the mechanisms of neuronal injury and neuronal cell loss remain unclear. The aim of the present study was to examine the neural consequences of distal colitis and to assess the role of neutrophil granulocytes in mediating these changes. Colitis was induced in C3H/HEN female mice with dinitrobenzene sulfonic acid. The mice were then sacrificed at 0.5, 1, 1.5, 2, 3, 4, 6, 12, 24, 120 h post instillation of dinitrobenzene sulfonic acid. The inflammatory response was assessed by macroscopic damage score, myeloperoxidase activity and histology. HuC/D and PGP 9.5 immunostaining was used to examine myenteric plexus density and structure, neural cell body numbers and distribution in cross-section and whole mount preparations. Apoptosis was investigated in whole mount preparations double stained with HuC/D and activated caspase-3 or cleaved poly (ADP-ribose) polymerase (PARP). Dinitrobenzene sulfonic acid-induced colitis was associated with a rapid and significant loss of HuC/D immunoreactive myenteric plexus neuronal cell bodies (42% decrease relative to control) that remained unchanged between 6 and 120 h. No change in myenteric plexus density was observed with PGP 9.5 immunostaining. Neuronal apoptosis was evident between 0.5 and 3 h. PARP immunoreactive neurons ranged between 1% and 2.5%. Colitis was associated with significant impairment in colonic propulsive function. Pre-treatment of mice with anti-neutrophil serum attenuated the inflammatory response and partially reduced the extent of myenteric plexus neuronal cell loss. Taken together, these data suggest that acute colitis is associated with loss of myenteric plexus neurons that is partly mediated by neutrophil granulocyte infiltration and is accompanied by impairment of colonic motility.     

Recording ionic events from cultured, DiI-labelled myenteric neurons in the guinea-pig proximal colon

To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular. Neurons labeled after DiI was applied to circular muscle, fired in either a phasic or a tonic manner, and exhibited fast afterhyperpolarizations (100-300 ms duration) at the end of a depolarizing pulse. They expressed a fast inward current and at least three different outward currents. Action potentials elicited in DiI-labeled sensory neurons were followed by a prolonged afterhyperpolarization (AH, 4-6 s). The offset of a suprathreshold depolarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane depolarization in DiI-labeled neurons loaded with fura-2, robust Ca(2+) transients were recorded using the perforated patch technique. These results demonstrate that DiI labeling of cultured myenteric neurons is feasible, and patch clamp/Ca(2+) fluorescence recordings can be made from specific populations of cultured DiI-labeled colonic myenteric neurons.     

Excitatory actions of motilin on myenteric neurons of the guinea-pig small intestine

Motilin is considered as a key factor in controlling interdigestive migrating contractions. The present electrophysiological experiments were performed in vitro to examine actions of motilin on myenteric neurons of guinea-pigs after 18-h fasting period. Superfusion of motilin depolarized both S and AH neurons; the lowest effective concentration was 10 nM, and motilin depolarization was observed in 9 of 23 S neurons and in 5 of 25 AH neurons. The motilin depolarizations were associated with an increase in neuronal input resistance. The motilin responses were preserved in Ca2+ free/high Mg2+ solution in which no Ca2+ dependent synaptic transmission occurred. The reversal potential of the motilin responses was estimated about -95 mV, close to the equilibrium potential for K+. Furthermore, muscarinic depolarizations were blocked during the motilin responses. All of these indicated that motilin directly excited myenteric neurons mainly by inactivating K+ channels. It is concluded that motilin might modulate neuronal excitability of the myenteric plexus, leading to the control of interdigestive migrating contractions.     

R‐type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

Background R‐type Ca²⁺ channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown. Methods In the present study, we used intracellular electrophysiological techniques to determine the function of R‐type Ca²⁺ channels in myenteric neurons in the acutely isolated longitudinal muscle‐myenteric plexus. We used immunohistochemical methods to localize the Ca_V2.3 subunit of the R‐type Ca²⁺ channel in myenteric neurons. We also studied the effects of the non‐selective Ca²⁺ channel antagonist, CdCl₂ (100 μmol L^?1), the R‐type Ca²⁺ channel blockers NiCl₂ (50 μmol L^?1) and SNX‐482 (0.1 μmol L^?1), and the N‐type Ca²⁺ channel blocker ω‐conotoxin GVIA (CTX 0.1 μmol L^?1) on action potentials and fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs) in S and AH neurons in vitro. Key Results Ca_V2.3 co‐localized with calretinin and calbindin in myenteric neurons. NiCl₂ and SNX‐482 reduced the duration and amplitude of action potentials in AH but not S neurons. NiCl₂ inhibited the afterhyperpolarization in AH neurons. ω‐conotoxin GVIA, but not NiCl₂, blocked sEPSPs in AH neurons. NiCl₂ and SNX‐482 inhibited cholinergic, but not cholinergic/purinergic, fEPSPs in S neurons. Conclusions and Inferences These data show that R‐type Ca²⁺ channels contribute to action potentials, but not slow synaptic transmission, in AH neurons. R‐type Ca²⁺ channels contribute to release of acetylcholine as the mediator of fEPSPs in some S neurons. These data indicate that R‐type Ca²⁺ channels may be a target for drugs that selectively modulate activity of AH neurons or could alter fast synaptic excitation in specific pathways in the myenteric plexus.