Search

Background & Aims: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells)., Methods: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function., Results: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings., Conclusions: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll-like receptors (TLRs) in mice., Methods: We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creER T2 :tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor. We performed whole gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S ribosomal RNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creER T2 :tdTomato mice and incubated with agonists of TLR2 (receptor for gram-positive bacteria), TLR4 (receptor for gram-negative bacteria), or distilled water (control) and analyzed by flow cytometry., Results: Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of gram-positive bacteria and increased abundance of gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced neuronal nitric oxide synthase production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive enteric neural precursor cells expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from enteric neural precursor cells to approximately 10%, compared with approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice., Conclusions: In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via a specific signaling pathway. Pharmacologic and probiotic approaches directed towards specific TLR2 signaling processes might be developed for treatment of colonic motility disorders related to use of antibiotics or other factors., (Copyright © 2020. Published by Elsevier Inc.)

Background & Aims: A dysregulated response of CD4(+) T cells against the microbiota contributes to the development of inflammatory bowel disease. Effector CD4(+) T cells, generated in response to microbe-derived antigens, can reduce somatic inflammatory pain through the local release of opioids. We investigated whether colitogenic CD4(+) T cells that accumulate in the inflamed colon also produce opioids and are able to counteract inflammation-induced visceral pain in mice., Methods: Colitis was induced via transfer of naive CD4(+)CD45RB(high) T cells to immune-deficient mice or by administration of dextran sulfate sodium. Mice without colitis were used as controls. Samples of colon tissue were collected, and production of opioids by immune cells from inflamed intestine was assessed by quantitative polymerase chain reaction and cytofluorometry analyses. The role of intestinal opioid tone in inflammation-induced visceral hypersensitivity was assessed by colorectal distention., Results: In mice with T cell- or dextran sulfate sodium-induced colitis, colitogenic CD4(+) T cells (T-helper 1 and Th17 cells) accumulated in the inflamed intestine and expressed a high level of endogenous opioids. In contrast, macrophages and epithelial cells did not express opioids; opioid synthesis in the myenteric plexus was not altered on induction of inflammation. In mice with colitis, the local release of opioids by colitogenic CD4(+) T cells led to significant reduction of inflammation-associated visceral hypersensitivity., Conclusions: In mice, colitogenic Th1 and Th17 cells promote intestinal inflammation and colonic tissue damage but have simultaneous opioid-mediated analgesic activity, thereby reducing abdominal pain., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Glial cell-derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system during embryogenesis. We have observed the presence of Gdnf transcripts in the gastrointestinal tract of adult mice, and its early up-regulation after inflammation. We therefore investigated the effects of GDNF on enteric neuronal function in vitro., Methods: Primary neuronal cultures were established from isolated myenteric plexi, and characterized by immunostaining and Ca(2+) imaging. Gene expression of several ion channels was analyzed by quantitative polymerase chain reaction (PCR) and the electrophysiologic properties of the neurons were studied by patch clamp., Results: GDNF enhanced synaptogenesis and intercellular communication in primary myenteric neuronal cultures. Expression profiling revealed that GDNF exposure results in an up-regulation of Htr3a expression in the cultures and a similar increase was observed in inflamed colonic tissue where Gdnf expression was also increased. The increased Htr3a expression was accompanied by a functional increase in the response of neurons to acute challenge with 5-hydroxytryptamine (5-HT). GDNF treatment also caused inhibition of delayed rectifying voltage-gated potassium (Kv) currents, which correlated with the up-regulation of Htr3a and 5-HT-induced responses. Furthermore, pharmacologic blockade of Kv channels mimicked the effect of GDNF by increasing Htr3a expression as well as enhancing 5-HT-induced responses in the cultured myenteric neurons., Conclusions: GDNF promotes synaptic communication in cultured myenteric neurons. It also up-regulates 5-HT(3a)-receptor expression via modulation of Kv channel activity. Up-regulation of Gdnf after gastrointestinal inflammation might play an important role in the pathophysiology of gastrointestinal diseases., (2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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.

Background & Aims: In human and canine colon, both slow (slow waves, 2-8/min) and fast (myenteric potential oscillations [MPOs]; 16-20/min) electrical rhythms in the smooth muscle originate at the submucosal and myenteric borders, respectively. We used Ca(2+) imaging to investigate whether interstitial cells of Cajal (ICCs) at these borders generated distinct rhythms., Methods: Segments of canine colon were pinned with submucosal or myenteric surface uppermost or cut in cross section. Tissues were loaded with a Ca(2+) indicator (fluo-4), and activity was monitored at 36.5 +/- 0.5 degrees C using an electron multiplying charge coupled device (EMCCD)., Results: Rhythmic, biphasic Ca(2+) transients (5-8/min), similar in waveform to electrical slow waves, propagated without decrement as a wave front (2-5 mm/s) through the ICC-SM network lying along the submucosal surface of the circular muscle (CM). In contrast, rhythmic intracellular Ca(2+) waves (approximately 16/min) and spontaneous reductions in Ca(2+) were observed in ICCs at the myenteric border (ICC-MY). Normally, intracellular Ca(2+) waves were unsynchronized between adjacent ICC-MY, although excitatory nerve activity synchronized activity. In addition, spontaneous reductions in Ca(2+) were observed that inhibited Ca(2+) waves. N omega-nitro-L-arginine (100 micromol/L; nitric oxide antagonist) blocked the reductions in Ca(2+) and increased the frequency (approximately 19/min) of intracellular Ca(2+) waves within ICC-MY., Conclusions: ICC-SMs form a tightly coupled network that is able to generate and propagate slow waves. In contrast, Ca(2+) transients in ICC-MYs, which are normally not synchronized, have a similar duration and frequency as MPOs. Like MPOs, their activity is inhibited by nitrergic nerves and synchronized by excitatory nerves.

Background & Aims: Enteric glia are intimately associated with neurons in the enteric nervous system (ENS) and display morphologic and molecular similarities to central nervous system (CNS) astrocytes. Enteric glia express neurotransmitter receptors, suggesting that, like astrocytes, they are active participants in neuronal communication. In the ENS, the purine adenosine triphosphate (ATP) is co-released with the neurotransmitters noradrenaline and acetylcholine. Enteric glia express purinergic receptors and respond to ATP in vitro, suggesting that enteric glia participate in functional gastrointestinal responses to nerve signaling. We investigated whether enteric glia are activated by ATP released from enteric neurons., Methods: Synaptic activity was elicited in enteric neurons by electrically stimulating interganglionic connectives in the myenteric plexus of the guinea pig colon. Activity in enteric glial cells was detected by imaging intracellular calcium in situ., Results: Neuronal stimulation elicited increases in intracellular calcium in enteric glial cells that were blocked by tetrodotoxin, the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), and the phospholipase C inhibitor U73122. Furthermore, enteric glia responded robustly to exogenously applied ATP in situ, and the ATP response was blocked by PPADS and U73122. Data from pharmacologic profiling and immunohistochemical analyses support the hypothesis that P2Y4 is the major functional receptor underlying the ATP response in enteric glia., Conclusions: Our results provide direct evidence for functional purinergic neuron-glia communication in the enteric nervous system, raising the possibility that ATP released with neurotransmitters during enteric synaptic transmission functions to signal to enteric glia.

Background & Aims: Like the heart, intestinal smooth muscles exhibit electrical rhythmicity, which originates in pacemaker cells surrounding the myenteric plexus, called interstitial cells of Cajal (ICC-MY). In large mammals, ICC also line septa (ICC-SEP) between circular muscle (CM) bundles, suggesting they might be necessary for activating muscle bundles. It is important to determine their functional significance, because a loss of ICC in humans is associated with disordered motility. Our aims were therefore to determine the role of ICC-SEP in activating the thick CM in the human jejunum., Methods: The mucosa and submucosa were removed and muscle strips were cut and pinned in cross-section so that the ICC-MY and ICC-SEP networks and the CM could be readily visualized. The ICC networks and CM were loaded with the Ca(2+) indicator fluo-4, and pacemaker and muscle activity was recorded at 36.5 +/- 0.5( degrees )C., Results: Ca(2+) imaging revealed that pacemaker activity in human ICC-MY can entrain ICC-SEP to excite CM bundles. Unlike the heart, pacemaker activity in ICC-MY varied in amplitude, propagation distance, and direction, leading to a sporadic activation of ICC-SEP., Conclusions: ICC-SEP form a crucial conduction pathway for spreading excitation deep into muscle bundles of the human jejunum, necessary for motor patterns underlying mixing. A loss of these cells could severely affect motor activity.

Background & Aims: It has been generally assumed that interstitial cells of Cajal (ICC) in the human gastrointestinal tract have similar functions to those in rodents, but no direct experimental evidence exists to date for this assumption. This is an important question because pathologists have noted decreased numbers of ICC in patients with a variety of motility disorders, and some have speculated that loss of ICC could be responsible for motor dysfunction. Our aims were to determine whether myenteric ICC (ICC-MY) in human jejunum are pacemaker cells and whether these cells actively propagate pacemaker activity., Methods: The mucosa and submucosa were removed, and strips of longitudinal muscle were peeled away to reveal the ICC-MY network. ICC networks were loaded with the Ca(2+) indicator fluo-4, and pacemaker activity was recorded via high-speed video imaging at 36.5 degrees C +/- 0.5 degrees C., Results: Rhythmic, biphasic Ca(2+) transients (6.03 +/- 0.33 cycles/min) occurred in Kit-positive ICC-MY. These consisted of a rapidly propagating upstroke phase that initiated a sustained plateau phase, which was associated with Ca(2+) spikes in neighboring smooth muscle. Pacemaker activity was dependent on inositol 1,4,5-triphosphate receptor-operated stores and mitochondrial function. The upstroke phase of Ca(2+) transients in ICC-MY appeared to result from Ca(2+) influx through dihydropyridine-resistant Ca(2+) channels, whereas the plateau phase was attributed to Ca(2+) release from inositol 1,4,5-triphosphate receptor-operated Ca(2+) stores., Conclusions: Each ICC-MY in human jejunum generates spontaneous pacemaker activity that actively propagates through the ICC network. Loss of these cells could severely disrupt the normal function of the human small intestine.

Background & Aims: Transit of fecal material through the human colon takes > or =30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena., Methods: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca(2+) imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit., Results: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-omega-nitro-L-arginine (100 micromol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca(2+) imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS +ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N-omega-nitro-L-arginine (100 micromol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility., Conclusions: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS +ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.

Background & Aims: Glial-derived neurotrophic factor (GDNF) promotes the survival and proliferation of enteric neurons. Neuropeptide Y (NPY) is an important peptide regulating gastrointestinal motility. The role of NPY on the survival and proliferation of enteric neurons is not known. We examined the effects of GDNF on the expression and release of NPY from enteric neurons and the role of NPY in promoting enteric neuronal proliferation and survival., Methods: Studies were performed in primary enteric neuronal cultures and NPY knockout mice (NPY(-/-)). GDNF-induced expression of NPY was assessed by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay. Using NPY-siRNA and NPY-Y1 receptor antagonist, we examined the role of NPY in mediating the survival and proliferation effects of GDNF. Gastrointestinal motility was assessed by measuring gastric emptying, intestinal transit, and isometric muscle recording from intestinal muscle strips., Results: GDNF induced a significant increase in NPY messenger RNA and protein expression in primary enteric neurons and the release of NPY into the culture medium. NPY (1 mumol/L) significantly increased proliferation of neurons and reduced apoptosis. In the presence of NPY-siRNA and NPY-Y1 receptor antagonist or in enteric neurons cultured from NPY(-/-) mice, GDNF-mediated neuronal proliferation and survival was reduced. NPY increased the phosphorylation of Akt, a downstream target of the PI-3-kinase pathway. In NPY(-/-) mice, there were significantly fewer nNOS-containing enteric neurons compared with wild-type (WT) mice. NPY(-/-) mice had accelerated gastric emptying and delayed intestinal transit compared with WT mice., Conclusions: We demonstrate that NPY acts as an autocrine neurotrophic factor for enteric neurons.

Background & Aims: Serine proteases are postulated to influence gastrointestinal function by stimulating protease-activated receptors (PARs). This study identified the effects on myenteric neurons of activating PARs and investigated underlying mechanisms of action., Methods: Intracellular electrophysiologic methods were used to study the effects of proteases on electrical and synaptic behavior of morphologically identified neurons in the guinea pig enteric nervous system. Fluorescent immunohistochemistry was used to study the chemical coding of neurons that responded to PARs stimulation., Results: Application of thrombin, trypsin, or mast cell tryptase evoked slowly activating excitatory responses reminiscent of slow synaptic excitation in enteric neurons. Synthetic activating peptides for PAR-1, -2, and -4 receptors mimicked the actions of the proteases. The depolarizing responses evoked by PARs were insensitive to cyclooxygenase inhibitors and were suppressed by agents that inhibit phospholipase C (PLC) or block intraneuronal receptors for inositol triphosphate. A majority of PAR-sensitive uniaxonal neurons expressed immunoreactivity for nitric oxide synthase. Most of the PAR-sensitive AH Dogiel morphologic type II neurons were immunoreactive for calbindin., Conclusions: Excitatory responses to the serine proteases are mediated by PAR-1, -2, and -4 receptors. The mechanism of signal transduction involves stimulation of PLC and intraneuronal calcium mobilization and is independent of prostanoid formation.

Background & Aims: Interstitial cells of Cajal (ICC) serve as pacemaker cells and mediators of neurotransmission from the enteric nervous system to gastrointestinal muscles. ICC develop from mesenchymal cells that express c-Kit, and signaling via Kit receptors is necessary for normal development of ICC. We studied the fate of functionally developed ICC after blockade of Kit receptors to determine whether ICC undergo cell death or whether the phenotype of the cells is modified. The fate of undeveloped ICC was also investigated., Methods: Neutralizing, anti-Kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. ICC in the small intestine were examined by immunohistochemistry and electron microscopy. Occurrence of apoptosis was also assayed., Results: When Kit receptors were blocked, ICC nearly disappeared from the small intestine. Apoptosis was not detected in regions where ICC are normally distributed. Remaining Kit-immunopositive cells in the pacemaker region of the small intestine developed ultrastructural features similar to smooth muscle cells, including prominent filament bundles and expression of the muscle-specific intermediate filament protein, desmin, and smooth muscle myosin. ICC of the deep muscular plexus normally develop after birth in the mouse. Precursors of these cells remained in an undifferentiated state when Kit was blocked., Conclusions: These data, along with previous studies showing that ICC in the pacemaker region of the small intestine and longitudinal muscle cells develop from the same Kit-immunopositive precursor cells, suggest inherent plasticity between the ICC and smooth muscle cells that is regulated by Kit-dependent cell signaling.

Background & Aims: Adenosine triphosphate (ATP) acting at P2 receptors mediates some fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons of guinea pig ileum. The present studies investigate the distribution of purinergic fEPSPs along the length of the gut and characterize the P2-receptor subtype mediating fEPSPs., Methods: Conventional intracellular electrophysiological methods were used to record from myenteric neurons in vitro., Results: At a membrane potential of -97 +/- 1 mV, the amplitude (25 +/- 1 mV; n = 307) of fEPSPs was similar along the gut. Hexamethonium (100 micromol/L) inhibited fEPSPs in the gastric corpus by 98% +/- 1% (n = 31) and in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 42%-55%. In the presence of hexamethonium, suramin (100 micromol/L) or the P2X antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 micromol/L) reduced the control fEPSP amplitude in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 71%-84%. The pharmacology of the purinergic fEPSPs was investigated in detail in the ileum. Noncholinergic fEPSPs were concentration-dependently (1-30 micromol/L) inhibited by PPADS (50%-inhibitory concentration, 3 micromol/L). In addition, alpha,beta-methylene 5'-adenosine triphosphate (1 micromol/L) also reduced purinergic fEPSPs., Conclusions: Fast EPSPs mediated in part through P2X receptors are prominent in myenteric neurons along the small and large intestines but are rare in the gastric corpus.

Background & Aims: Peristaltic contraction of the esophagus and relaxation of the lower esophageal sphincter (LES) are mediated by nitric oxide (NO.)-producing myenteric nerves. NO synthase immunoreactivity and calcitonin gene-related peptide (CGRP) immunoreactivity colocalize in these nerves. CGRP relaxes the LES and decreases the amplitude of esophageal peristalsis. The aim of this study was to test the hypothesis that CGRP plays a role in nerve-induced responses of the esophagus., Methods: Smooth muscle strips from the LES and the body of the esophagus were stimulated by an electrical field to evoke nerve-mediated responses., Results: CGRP decreased the amplitude and lengthened the latency of the off-response, and it relaxed the LES. Tetrodotoxin did not block the CGRP-induced LES relaxation. The CGRP antagonist CGRP 8-37 inhibited the effects of exogenous CGRP. It increased the amplitude and shortened the latency of the off-response, and it increased the resting tone of the LES. CGRP 8-37 did not inhibit LES relaxation or esophageal contraction. N omega-nitro-L-arginine abolished nerve-induced relaxation of the LES muscle and the esophageal off-response but not the relaxation produced by CGRP., Conclusions: These data support the hypothesis that CGRP may decrease the LES basal tone and modulate the timing and amplitude of the esophageal off-responses.

Background/aims: Long-term myenteric and extrinsic denervation of a segment of rat jejunum results in increased stress generation by the longitudinal muscle layer of the denervated segment 15 days after denervation. This study examined whether alterations in the properties of either cell membrane calcium channels and/or sarcoplasmic reticular Ca(2+)-adenosine triphosphatase (ATPase) contribute to the increased stress development., Methods: The effects of the calcium channel blocker nifedipine and the sarcoplasmic reticular Ca(2+)-ATPase inhibitor cyclopiazonic acid on the contractile activity of denervated and control smooth muscle were determined., Results: The ability of nifedipine to inhibit KCl-induced contractions was significantly increased in denervated tissues; however, there was no difference in the potency of nifedipine when tissues were stimulated with carbachol. Calcium concentration-response curves obtained in the presence of either KCl or carbachol were determined in tissues previously depleted of calcium. Long-term denervated tissues showed an increased sensitivity to calcium and a decreased maximum contractile response after stimulation with carbachol. Cyclopiazonic acid inhibited repletion of intracellular calcium stores of control muscle but had no effect in denervated tissue., Conclusions: Long-term denervation of a segment of rat small intestine results in profound alterations in calcium metabolism at the cell membrane and, to a lesser extent, at the sarcoplasmic reticulum of smooth muscle cells of the longitudinal muscle layer.

Background/aims: Recent studies with neurofilament antibodies as neuronal markers have shown subpopulations of myenteric neurons that do not contain neurofilament proteins. Novel neuronal intermediate filament proteins alpha-internexin, peripherin, and nestin have been identified. The aim of this study was to examine the distribution of these novel intermediate filaments in comparison with neurofilaments in myenteric plexus neurons., Methods: Using indirect immunofluorescence techniques in whole-mount cryostat sections from neonate and adult rat small intestine and in primary cultures of myenteric neurons, the distribution of neurofilaments, alpha-internexin, peripherin, and nestin was studied in comparison with the neuronal marker protein gene product (PGP) 9.5 in myenteric neurons., Results: Sixty-five percent of neurons contained neurofilament triplet proteins. alpha-Internexin and/or peripherin were found in the neurofilament-negative neurons. PGP 9.5 was present in 80% of the myenteric neurons. Of the neurons that were PGP negative, > 95% contained peripherin or alpha-internexin. Nestin was not found in either neonate or adult myenteric neurons but was seen in glial cells in culture., Conclusions: The results suggest that a subpopulation of myenteric neurons lacks neurofilament triplet proteins but contains either peripherin, alpha-internexin, or both. This selective distribution of intermediate filaments in subpopulations of enteric neurons may support differential roles in these structurally unique neurons.

Background: The enteric nervous system is responsible in vivo for most of the change in fluid transport induced by cholera toxin. The aim of the present study was to investigate the importance of the myenteric plexus in the Intramural reflex responsible for this secretion., Methods: Long-term ablation of the myenteric plexus was achieved by serosal application of benzalkonium chloride on jejunal segments in rats., Results: The treated segments without functioning myenteric plexus showed a normal net fluid absorption. Cholera toxin in this segment only induced a reduction of fluid absorption, whereas in a nontreated ileal segment it concomitantly induced a conspicuous net fluid secretion. Intravenous hexamethonium did not change the cholera toxin response in the treated jejunal segments, whereas vasoactive intestinal polypeptide elicited a marked secretion., Conclusions: Benzalkonium chloride treatment eliminated the ability of cholera toxin to induce intestinal secretion. Thus, all afferent fibers in the intramural secretory reflex activated by cholera toxin are probably conveyed via the myenteric plexus, which functions as the integrating center in the enteric nervous system. The Ussing chamber technique using stripped intestinal preparations cannot be used when studying effects of luminal secretagogues.

Background: Adenosine acts at A1 receptors to inhibit the release of most neurotransmitters. This study tested the hypothesis that both exogenous adenosine (ADO) and tonic release of endogenous ADO act at presynaptic A1 receptors to suppress excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in myenteric neurons., Methods: Intracellular microelectrodes were used to study actions of ADO, the agonists 2-chloro-N6-cyclopentyl ADO, its 1-deaza derivative, 5'-N-ethylcarboxamido ADO, and CGS 21680 or the antagonists 8-cyclopentyl-1,3-dimethylxanthine, its 1,3-dipropyl analog, and 1,3-dipropyl-8-p-sulfophenylxanthine on synaptic behavior in myenteric neurons., Results: Each of the agonists suppressed slow EPSPs in all 35 AH/type 2, 8 of 10 S/type 1, and 7 of 7 nonspiking neurons. ADO also decreased neuronal excitability (n = 63) in AH/type 2 neurons. Agonists suppressed fast nicotinic EPSPs in all 20 S/type 1, 10 nonspiking, and 3 AH/type 2 neurons without having any effect on postsynaptic responses to nicotinic agonists. CCPA was more potent than CGS 21680 in suppressing EPSPs. In 30% of neurons, the only action of antagonists was to block the effect of A1 or A2 agonists on EPSPs. Agonists did not inhibit IPSPs, but unmasked robust slow IPSPs by preventing slow EPSPs. Antagonists acted alone to enhance EPSPs in 70% of neurons., Conclusions: (1) ADO acts at presynaptic A1 sites to suppress EPSPs in all neurons, (2) IPSPs are revealed by ADO, and (3) ongoing release of endogenous ADO inhibits synaptic transmission.

We have quantified neurotransmitter changes in the rat jejunum in which the myenteric neurons were ablated by serosal application of benzalkonium chloride. Within 2 days after benzalkonium chloride treatment, there was a 40% reduction in the activity of choline acetyltransferase, a specific marker for cholinergic neurons, and a 25% reduction in the amount of vasoactive intestinal peptide per centimeter length of jejunum. By 15 days, levels were comparable to those in control segments, and by 45 days after myenteric neuronal ablation, levels in treated tissues were twice those in controls. In contrast, we observed no reduction in the amount of leucine-enkephalin per centimeter length of jejunum at early times after benzalkonium chloride treatment, although by 45 days, levels of this neurotransmitter in treated segments of jejunum were more than twice those in controls. Significant increases in muscle thickness and tissue weight were also observed at 15, 30, and 45 days after myenteric neuronal ablation. Thus we have observed that in response to chemical ablation of myenteric neurons in the rat jejunum, there is a thickening of the smooth muscle layers and a compensatory increase in the production of certain neurotransmitters by the surviving neuronal elements in the gut.

Norepinephrine, acetylcholine, and certain peptides are contained in mucosal nerves and have potent effects on transepithelial water and electrolyte fluxes. It is difficult to ascribe roles for these nerves as their sources are unknown. The present studies were undertaken to determine the origins of nerve fibers that are found in the mucosa of the guinea pig small intestine and which contain one of the following substances: vasoactive intestinal peptide, substance P, somatostatin, neuropeptide Y, cholecystokinin, or norepinephrine. Nerve fiber origins were ascertained by making lesions to sever pathways through which the nerves could reach the mucosa. The lesioning operations were homotopic autotransplants of short (2 cm) segments of intestine; myectomies, in which a 5-10-mm length of intestine was stripped of longitudinal muscle and myenteric plexus; and extrinsic denervation, in which nerves reaching the intestine through the mesentery were severed. The results of these studies, considered along with previously published work, led to the upcoming conclusions. Nerve fibers in the mucosa showing immunoreactivity for vasoactive intestinal peptide, somatostatin, cholecystokinin, and neuropeptide Y arise from cell bodies in the overlying submucous plexus. Substance P fibers arise in part from the overlying submucous plexus and in part from the overlying myenteric plexus. Mucosal norepinephrine fibers arise from extrinsic sympathetic ganglia. Enkephalin, gastrin-releasing peptide, and 5-hydroxytryptamine, which are in some enteric nerves, are not found in submucous nerve cells and few, if any, fibers containing these substances supply the mucosa. Thus, the mucosa receives a dense nerve supply, much of which arises locally from submucous ganglia.

Serosal application of benzalkonium chloride (BAC), a cationic surfactant, was used to selectively ablate the myenteric neurons of the rat jejunum. The myoelectric activity of the BAC-treated area and the areas both orad and caudad to it were assessed. In the jejunal segment devoid of myenteric neurons, the basic electric rhythm (BER) pattern was erratic and the amplitude of the BER was attenuated. The BER frequency of the jejunal area caudad to the BAC-treated area was 30.7 +/- 0.73 cycles/min, which was significantly reduced from the normal BER frequency (37.7 +/- 0.64 cycles/min). Spike activity was normal in areas both orad and caudad to the treated area, existing in regular cycling bursts (migrating myoelectric complex). Spiking in the treated area did not appear until approximately 13-17 days after BAC treatment. These spikes, though of greater duration and lower frequency than the adjacent areas, always appeared after an orad burst and were followed by a caudad burst. In addition, the following motility parameters of the regions orad and caudad to the treated area were not significantly different from control animals: burst duration, period, and burst propagation velocity. In conclusion, the present investigation has demonstrated that BAC-induced ablation of the myenteric neurons in the rat jejunum disrupts the BER but not the migrating myoelectric complex propagation. This suggests that the myenteric neurons play a modulatory role in the generation and propagation of the BER, whereas humoral factors or the submucosal neurons may be more important in the control of the migrating myoelectric complex.

The effects of surgical interruption of the myenteric plexus (myectomy), extrinsic denervation of a length of small intestine, or transection and reanastomosis of the intestinal wall on migration of phase III of the migrating myoelectric complex was studied in guinea pigs. In addition, the recovery of phase III migration and the regrowth of intestinal nerves and muscle across the reanastomosis was studied at various times up to 60 days after surgery. At 6-9 days after surgery, phase III did not migrate past the myectomy during 50%-60% of recorded migrating myoelectric complexes and transection and reanastomosis of the intestinal wall blocked aboral progression of phase III in 90% of cases. Extrinsic denervation did not alter phase III migration through the denervated segment. Phase III migration past the reanastomosis recovered with time after surgery; 80% recovery occurred by 60 days after surgery. Immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin were used as markers for intestinal nerves that were cut by transaction. Immunoreactivities for vasoactive intestinal peptide and gastrin-releasing peptide are contained in myenteric neurons that project in an oral to anal direction to other myenteric ganglia and to the circular muscle. Immunoreactivity for somatostatin is contained in nerve fibers projecting aborally to other myenteric ganglia. At 7-15 days after surgery, there were accumulations of immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin in nerve fibers on the oral side of the reanastomosis, but nerve fibers containing these peptides were not observed in myenteric ganglia or circular muscle close to the anal edge. At 23-28 days, immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin nerve fibers were traced across the reanastomosis and nerve terminals were detected in ganglia and muscle close to the lesion on the anal side. Nerve fibers traversed the lesion in all cases at 57-60 days and vasoactive intestinal peptide-, gastrin-releasing peptide-, and somatostatin-immunoreactive nerve terminals were detected in the first two to three rows of myenteric ganglia on the anal side. Regrowth of intestinal muscle followed a similar time-course to that observed for nerves. These data suggest that interruption of the myenteric plexus alone does not completely block phase III migration. In addition, recovery of phase III migration past a reanastomosis is associated with a restoration of both nervous and mechanical connections.