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Alternative nutrient sources to fishmeal for fish feed, such as insect meals, represent a promising sustainable supply. However, the consequences for fish digestive function have not been exhaustively investigated. In the present study we evaluated the effect of partial fishmeal substitution with 10% Hermetia illucens (Hi10) larvae meal on the neuromuscular function of proximal and distal intestine in gilthead sea bream. In animals fed with insect meal, weight and growth parameters were similar to controls fed with conventional fishmeal. In addition, no anomalies in intestinal gross morphology and no overt signs of inflammation were observed. The gastrointestinal transit was significantly reduced in Hi10 fed animals. In the proximal and distal intestine longitudinal muscle, Hi10 feeding downregulated the excitatory cholinergic and serotoninergic transmission. Sodium nitroprusside-induced inhibitory relaxations increased in the proximal intestine and decreased in the distal intestine after Hi10 meal. Changes in the excitatory and inhibitory components of peristalsis were associated with adaptive changes in the chemical coding of both proximal and distal intestine myenteric plexus. However, these neuromuscular function alterations were not associated with considerable variations in morphometric growth parameters, suggesting that 10% Hi meal may represent a tolerable alternative protein source for gilthead sea bream diets.

Antidopaminergic gastrointestinal prokinetics are indeed commonly used to treat gastrointestinal motility disorders, although the precise role of dopaminergic transmission in the gut is still unclear. Since dopamine transporter (DAT) is involved in several brain disorders by modulating extracellular dopamine in the central nervous system, this study evaluated the impact of DAT genetic reduction on the morpho-functional integrity of mouse small intestine enteric nervous system (ENS). In DAT heterozygous (DAT+/-) and wild-type (DAT+/+) mice (14 ± 2 weeks) alterations in small intestinal contractility were evaluated by isometrical assessment of neuromuscular responses to receptor and non-receptor-mediated stimuli. Changes in ENS integrity were studied by real-time PCR and confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations (). DAT genetic reduction resulted in a significant increase in dopamine-mediated effects, primarily via D1 receptor activation, as well as in reduced cholinergic response, sustained by tachykininergic and glutamatergic neurotransmission via NMDA receptors. These functional anomalies were associated to architectural changes in the neurochemical coding and S100β immunoreactivity in small intestine myenteric plexus. Our study provides evidence that genetic-driven DAT defective activity determines anomalies in ENS architecture and neurochemical coding together with ileal dysmotility, highlighting the involvement of dopaminergic system in gut disorders, often associated to neurological conditions. ispartof: BIOMEDICINES vol:9 issue:5 ispartof: location:Switzerland status: published

Inflammatory bowel diseases (IBDs) are a group of inflammatory conditions of the colon and small intestine, including Crohn's disease and ulcerative colitis. Since Danio rerio is a promising animal model to study gut function, we developed a soy‐dependent model of intestinal inflammation in adult zebrafish. The soya bean meal diet was given for 4 weeks and induced an inflammatory process, as demonstrated by morphological changes together with an increased percentage of neutrophils infiltrating the intestinal wall, which developed between the second and fourth week of treatment. Pro‐inflammatory genes such as interleukin‐1beta, interleukin‐8 and tumour necrosis factor alpha were upregulated in the second week and anti‐inflammatory genes such as transforming growth factor beta and interleukin‐10. Interestingly, an additional expression peak was found for interleukin‐8 at the fourth week. Neuronal genes, OTX1 and OTX2, were significantly upregulated in the first two weeks, compatible with the development of the changes in the gut wall. As for the genes of the p53 family such as p53, DNp63 and p73, a statistically significant increase was observed after two weeks of treatment compared with controls. Interestingly, DNp63 and p73 were shown an additional peak after four weeks. Our data demonstrate that soya bean meal diet negatively influences intestinal morphology and immunological function in adult zebrafish showing the features of acute inflammation. Data observed at the fourth week of treatment may suggest initiation of chronic inflammation. Adult zebrafish may represent a promising model to better understand the mechanisms of food‐dependent intestinal inflammation.

Background Oxidized phospholipid derivatives (OxPAPCs) act as bacterial lipopolysaccharide (LPS)-like damage-associated molecular patterns. OxPAPCs dose-dependently exert pro- or anti-inflammatory effects by interacting with several cellular receptors, mainly Toll-like receptors 2 and 4. It is currently unknown whether OxPAPCs may affect enteric nervous system (ENS) functional and structural integrity. Methods Juvenile (3 weeks old) male C57Bl/6 mice were treated intraperitoneally with OxPAPCs, twice daily for 3 days. Changes in small intestinal contractility were evaluated by isometric neuromuscular responses to receptor and non-receptor-mediated stimuli. Alterations in ENS integrity and serotonergic pathways were assessed by real-time PCR and confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs). Tissue levels of serotonin (5-HT), tryptophan, and kynurenine were measured by HPLC coupled to UV/fluorescent detection. Key results OxPAPC treatment induced enteric gliosis, loss of myenteric plexus neurons, and excitatory hypercontractility, and reduced nitrergic neurotransmission with no changes in nNOS+ neurons. Interestingly, these changes were associated with a higher functional response to 5-HT, altered immunoreactivity of 5-HT receptors and serotonin transporter (SERT) together with a marked decrease in 5-HT levels, shifting tryptophan metabolism toward kynurenine production. Conclusions and inferences OxPAPC treatment disrupted structural and functional integrity of the ENS, affecting serotoninergic tone and 5-HT tissue levels toward a higher kynurenine content during adolescence, suggesting that changes in intestinal lipid metabolism toward oxidation can affect serotoninergic pathways, potentially increasing the risk of developing functional gastrointestinal disorders during critical stages of development.

Intestinal ischemia/reperfusion (I/R) injury has severe consequences on myenteric neurons, which can be irreversibly compromised resulting in slowing of transit and hindered food digestion. Myenteric neurons synthesize hyaluronan (HA) to form a well-structured perineuronal net, which undergoes derangement when myenteric ganglia homeostasis is perturbed, i.e. during inflammation. In this study we evaluated HA involvement in rat small intestine myenteric plexus after in vivo I/R injury induced by clamping a branch of the superior mesenteric artery for 60 min, followed by 24 h of reperfusion. In some experiments, 4-methylumbelliferone (4-MU, 25 mg/kg), a HA synthesis inhibitor, was intraperitoneally administered to normal (CTR), sham-operated (SH) and I/R animals for 24 h. In longitudinal muscle myenteric plexus (LMMP) whole-mount preparations, HA binding protein staining as well as HA levels were significantly higher in the I/R group, and were reduced after 4-MU treatment. HA synthase 1 and 2 (HAS1 and HAS2) labelled myenteric neurons and mRNA levels in LMMPs increased in the I/R group with respect to CTR, and were reduced by 4-MU. The efficiency of the gastrointestinal transit was significantly reduced in I/R and 4-MU-treated I/R groups with respect to CTR and SH groups. In the 4-MU-treated I/R group gastric emptying was reduced with respect to the CTR, SH and I/R groups. Carbachol (CCh) and electrical field (EFS, 0.1–40 Hz) stimulated contractions and EFS-induced (10 Hz) NANC relaxations were reduced in the I/R group with respect to both CTR and SH groups. After I/R, 4-MU treatment increased EFS contractions towards control values, but did not affect CCh-induced contractions. NANC on-relaxations after I/R were not influenced by 4-MU treatment. Main alterations in the neurochemical coding of both excitatory (tachykinergic) and inhibitory pathways (iNOS, VIPergic) were also observed after I/R, and were influenced by 4-MU administration. Overall, our data suggest that, after an intestinal I/R damage, changes of HA homeostasis in specific myenteric neuron populations may influence the efficiency of the gastrointestinal transit. We cannot exclude that modulation of HA synthesis in these conditions may ameliorate derangement of the enteric motor function preventing, at least in part, the development of dysmotility.

Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.

Background and Purpose Gut microbiota is essential for the development of the gastrointestinal system, including the enteric nervous system (ENS). Perturbations of gut microbiota in early life have the potential to alter neurodevelopment leading to functional bowel disorders later in life. We examined the hypothesis that gut dysbiosis impairs the structural and functional integrity of the ENS, leading to gut dysmotility in juvenile mice. Experimental Approach To induce gut dysbiosis, broad-spectrum antibiotics were administered by gavage to juvenile (3weeks old) male C57Bl/6 mice for 14 days. Bile acid composition in the intestinal lumen was analysed by liquid chromatography-mass spectrometry. Changes in intestinal motility were evaluated by stool frequency, transit of a fluorescent-labelled marker and isometric muscle responses of ileal full-thickness preparations to receptor and non-receptor-mediated stimuli. Alterations in ENS integrity were assessed by immunohistochemistry and Western blot analysis. Key Results Antibiotic treatment altered gastrointestinal transit, luminal bile acid metabolism and bowel architecture. Gut dysbiosis resulted in distorted glial network, loss of myenteric plexus neurons, altered cholinergic, tachykininergic and nitrergic neurotransmission associated with reduced number of nNOS neurons and different ileal distribution of the toll-like receptor TLR2. Functional defects were partly reversed by activation of TLR2 signalling. Conclusions and Implications Gut dysbiosis caused complex morpho-functional neuromuscular rearrangements, characterized by structural defects of the ENS and increased tachykininergic neurotransmission. Altogether, our findings support the beneficial role of enteric microbiota for ENS homeostasis instrumental in ensuring proper gut neuromuscular function during critical stages of development.

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity.

Background Intestinal ischemia and reperfusion (I/R) injury leads to abnormalities in motility, namely delay of transit, caused by damage to myenteric neurons. Alterations of the nitrergic transmission may occur in these conditions. This study investigated whether an in vitro I/R injury may affect nitric oxide (NO) production from the myenteric plexus of the guinea pig ileum and which NO synthase (NOS) isoform is involved. Methods The distribution of the neuronal (n) and inducible (i) NOS was determined by immunohistochemistry during 60 min of glucose/oxygen deprivation (in vitro ischemia) followed by 60 min of reperfusion. The protein and mRNA levels of nNOS and iNOS were investigated by Western-immunoblotting and real time RT-PCR, respectively. NO levels were quantified as nitrite/nitrate. Key Results After in vitro I/R the proportion of nNOS-expressing neurons and protein levels remained unchanged. nNOS mRNA levels increased 60 min after inducing ischemia and in the following 5 min of reperfusion. iNOS-immunoreactive neurons, protein and mRNA levels were up-regulated during the whole I/R period. A significant increase of nitrite/nitrate levels was observed in the first 5 min after inducing I/R and was significantly reduced by Nω-propyl-l-arginine and 1400 W, selective inhibitors of nNOS and iNOS, respectively. Conclusions & Inferences Our data demonstrate that both iNOS and nNOS represent sources for NO overproduction in ileal myenteric plexus during I/R, although iNOS undergoes more consistent changes suggesting a more relevant role for this isoform in the alterations occurring in myenteric neurons following I/R.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by Nω-propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2.NEW & NOTEWORTHY Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.

The existence of a functional interplay between alpha(2)-adrenoceptor and opioid receptor inhibitory pathways modulating neurotransmitter release has been demonstrated in the enteric nervous system by development of sensitivity changes to alpha(2)-adrenoceptor, mu- and kappa-opioid receptor agents on enteric cholinergic neurons after chronic sympathetic denervation. In the present study, to further examine this hypothesis we evaluated whether manipulation of alpha(2)-adrenoceptor pathways by chronic treatment with the antidepressant drug, desipramine (10 mg/kg i.p. daily, for 21 days), could entail changes in enteric mu- and kappa-opioid receptor pathways in the myenteric plexus of the guinea pig distal colon. In this region, subsensitivity to the inhibitory effect of both UK14,304 and U69,593, respectively alpha(2A)-adrenoceptor and kappa-opioid receptor agonist, on the peristaltic reflex developed after chronic desipramine treatment. On opposite, in these experimental conditions, supersensitivity developed to the inhibitory effect of [D-Ala, N-Me-Phe4-Gly-ol5]-enkephalin (DAMGO), mu-opioid receptor agonist, on propulsion velocity. Immunoreactive expression levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly decreased in the myenteric plexus of the guinea pig colon after chronic desipramine treatment. In these experimental conditions, mRNA levels of alpha(2A)-adrenoceptors, mu- and kappa-opioid receptors significantly increased, excluding a direct involvement of transcription mechanisms in the regulation of receptor expression. Levels of G protein-coupled receptor kinase 2/3 and of inhibitory G(i/o) proteins were significantly reduced in the myenteric plexus after chronic treatment with desipramine. Such changes might represent possible molecular mechanisms involved in the development of subsensitivity to UK14,304 and U69,593 on the efficiency of peristalsis. Alternative molecular mechanisms, including a higher efficiency in the coupling between receptor activation and downstream intracellular effector systems, possibly independent from inhibitory G(i/o) proteins, may be accounted for the development of supersensitivity to DAMGO. Increased sensitivity to the mu-opioid agonist might compensate for the development of alpha(2A)-adrenoceptor and kappa-opioid receptor subsensitivity. On the whole, the present data further strengthen the concept that, manipulation of alpha(2)-adrenergic inhibitory receptor pathways in the enteric nervous system entails changes in opioid inhibitory receptor pathways, which might be involved in maintaining homeostasis as suggested for mu-opioid, but not for kappa-opioid receptors.

We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.

The actions of purine and pyrimidine compounds on isolated segments of the mouse intestine were investigated during postnatal development. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1,) P2X(2) and P2X(3) receptors were examined immunohistochemically, and levels of expression of P2Y(1), P2X(1) and P2X(2) were studied by Western immunoblot. From day 12 onwards, the order of potency for relaxation of longitudinal muscle of all regions was 2-MeSADPor=alpha,beta-meATPor=ATP=UTP=adenosine, suggesting P2Y(1) receptors. This was supported by the sensitivity of responses to 2-MeSADP to the selective antagonist MRS 2179 and P2Y(1) receptor immunoreactivity on longitudinal muscle and a subpopulation of myenteric neurons. A further alpha,beta-meATP-sensitive P2Y receptor subtype was also indicated. ATP and UTP were equipotent suggesting a P2Y(2) and/or P2Y(4) receptor. Adenosine relaxed the longitudinal muscle in all regions via P1 receptors. The efficacy of all agonists to induce relaxation of raised tone preparations increased with age, being comparable to adult by day 20, the weaning age. During postnatal development the contractile response of the ileum and colon was via P2Y(1) receptors, while the relaxant response mediated by P2Y(1) receptors gradually appeared along the mouse gastrointestinal tract, being detectable in the stomach from day 3 and in the duodenum from day 6. In the ileum and colon relaxant responses to 2-MeSADP were not detected until days 8 and 12, respectively. 2-MeSADP induced contractions on basal tone preparations from day 3, but decreased significantly at day 12 and disappeared by day 20. At day 8, contractions of colonic longitudinal muscle to ATP showed no desensitisation suggesting the involvement of P2X(2) receptors. Immunoreactivity to P2X(2) receptors only was observed on the longitudinal muscle of the colon and ileum from day 1 and on a subpopulation of myenteric neurons from day 3. These data suggest that P2Y(1) receptors undergo postnatal developmental changes in the mouse gut, with a shift from contraction to relaxation. Such changes occur 1 week before weaning and may contribute to the changes that take place in the gut when the food composition changes from maternal milk to solid food.

Chronic sympathetic denervation entails subsensitivity to alpha(2)-adrenoceptor agonists and supersensitivity to kappa- and mu-opioid receptor agonists modulating cholinergic neurons in the guinea pig colon. A possible role for signal transduction G proteins in contributing to development of these sensitivity changes was investigated. Pertussis toxin (PTX), a blocker of the G(i/o)-type family of G proteins significantly reduced the inhibitory effects of UK14,304 (alpha(2)-adrenoceptor agonist), U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on acetylcholine (ACh) overflow in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. In this experimental condition, immunoblot analysis revealed reduced levels of G(alphao), G(alphai2), G(alphai3) and G(beta) in myenteric plexus synaptosomes. On reverse, synaptosomal levels of G(alphai1) and G(alphaz), a PTX-insensitive G-protein, increased after chronic ablation of the sympathetic pathways. These data suggest that changes in the function and expression of inhibitory G proteins coupled to alpha(2)-adrenoceptors, kappa- and mu-opioid receptors occur in the myenteric plexus of the guinea pig colon after chronic sympathetic denervation. The possibility that regulation of G proteins represents one of the biochemical mechanisms at the basis of the changes in sensitivity of enteric cholinergic neurons to alpha(2)-adrenoceptor, kappa- and mu-opioid receptor agonists is discussed.

Alterations of the enteric glutamatergic transmission may underlay changes in the function of myenteric neurons following intestinal ischemia and reperfusion (I/R) contributing to impairment of gastrointestinal motility occurring in these pathological conditions. The aim of the present study was to evaluate whether glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R. Primary cultured rat myenteric ganglia were exposed to sodium azide and glucose deprivation (in vitro chemical ischemia). After 6 days of culture, immunoreactivity for NMDA, AMPA and kainate receptors subunits, GluN(1) and GluA(1-3), GluK(1-3) respectively, was found in myenteric neurons. In myenteric cultured ganglia, in normal metabolic conditions, -AP5, an NMDA antagonist, decreased myenteric neuron number and viability, determined by calcein AM/ethidium homodimer-1 assay, and increased reactive oxygen species (ROS) levels, measured with hydroxyphenyl fluorescein. CNQX, an AMPA/kainate antagonist exerted an opposite action on the same parameters. The total number and viability of myenteric neurons significantly decreased after I/R. In these conditions, the number of neurons staining for GluN1 and GluA(1-3) subunits remained unchanged, while, the number of GluK(1-3)-immunopositive neurons increased. After I/R, -AP5 and CNQX, concentration-dependently increased myenteric neuron number and significantly increased the number of living neurons. Both -AP5 and CNQX (100-500 µM) decreased I/R-induced increase of ROS levels in myenteric ganglia. On the whole, the present data provide evidence that, under normal metabolic conditions, the enteric glutamatergic system exerts a dualistic effect on cultured myenteric ganglia, either by improving or reducing neuron survival via NMDA or AMPA/kainate receptor activation, respectively. However, blockade of both receptor pathways may exert a protective role on myenteric neurons following and I/R damage. The neuroprotective effect may depend, at least in part, on the ability of both receptors to increase intraneuronal ROS production.

Enteric ganglia can maintain integrated functions, such as the peristaltic reflex, in the absence of input from the central nervous system, which has a modulatory role. Several clinical and experimental observations suggest that homeostatic control of gut function in a changing environment may be achieved through adaptive changes occurring in the enteric ganglia. A distinctive feature of enteric ganglia, which may be crucial during the development of adaptive responses, is the vicinity of the final effector cells, which are an important source of mediators regulating cell growth. The aim of this review is to focus on the possible mechanisms underlying neuronal plasticity in the enteric nervous system and to consider approaches to the study of plasticity in this model. These include investigations of neuronal connectivity during development, adaptive mechanisms that maintain function after suppression of a specific neural input, and the possible occurrence of activity-dependent modifications of synaptic efficacy, which are thought to be important in storage of information in the brain. One of the applied aspects of the study of plasticity in the enteric nervous system is that knowledge of the underlying mechanisms may eventually enable us to develop strategies to correct neuronal alterations described in several diseases. GASTROENTEROLOGY 1999;117:1438-1458

In the guinea-pig colon, acetylcholine (ACh) release from intrinsic cholinergic motor neurons is inhibited by adrenoceptors, opioid and muscarinic receptors. Chronic sympathetic denervation resulted in supersensitivity to the inhibitory effect of DAMGO (μ-opioid agonist) on ACh release and on the peristaltic reflex. After chronic treatment with naltrexone (NTX) supersensitivity to DAMGO and subsensitivity to UK14,304 (α2-adrenoceptor agonist) developed for both functional parameters. The facilitatory effect of scopolamine on ACh release remained unchanged after chronic NTX treatment, whereas it was potentiated after chronic sympathetic denervation. These data suggest the existence of a functional interaction between different inhibitory pathways modulating cholinergic motor neurons in the guinea-pig coloN. Namely, chronic manipulation of an inhibitory pathway may entail adaptive sensitivity changes in another inhibitory pathway so that homeostasis can be maintained.

We have studied the effect of [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, opioid mu-receptor agonist) and ICI-204,448 (kappa-receptor agonist) on endogenous noradrenaline release in the guinea-pig isolated distal colon. DAMGO enhances noradrenaline over-flow and this effect is antagonized by naloxone (pIC50 = 10.27) and nor-binaltorphimine (pIC50 = 7.97), and concentration-dependently turned into inhibition by yohimbine. ICI-204,448 inhibits noradrenaline overflow and is antagonized by naloxone (pIC50 = 9.38) and nor-binaltorphimine (pIC50 = 10.48), but is not affected by yohimbine. Evidence is thus given that mu- and kappa-opioid receptors modulate noradrenaline release in the guinea-pig colon. Modifications by yohimbine of the effect of DAMGO indicate the existence of a functional relationship between mu-receptors and alpha(2)-autoreceptors in this model.

In the guinea pig colon, chronic sympathetic denervation entails supersensitivity to inhibitory mu-opioid agents modulating cholinergic neurons. The mechanism underlying such adaptive change has not yet been unravelled, although protein kinase C (PKC) may be involved. A previous study indirectly demonstrated that activation of mu-opioid receptors on myenteric neurons facilitates PKC activity. Such coupling may counteract the inhibitory action of mu-opioid agents on acetylcholine overflow, since PKC, per se, increases this parameter. After chronic sympathetic denervation such restraint abates, representing a possible mechanism for development of supersensitivity to mu-opioid agents. In the present study, this hypothesis was further investigated. After chronic sympathetic denervation, Ca(2+)-dependent PKC activity was reduced in colonic myenteric plexus synaptosomes. The mu-opioid agent, DAMGO, increased Ca(2+)-dependent PKC activity in synaptosomes obtained from normal, but not from denervated animals. In myenteric synaptosomes obtained from this experimental group, protein levels of Ca(2+)-dependent PKC isoforms betaI, betaII and gamma decreased, whereas alpha levels increased. In whole-mount preparations, the four Ca(2+)-dependent PKC isoforms co-localized with mu-opioid receptors on subpopulations of colonic myenteric neurons. The percentage of neurons staining for PKCbetaII, as well as the number of mu-opioid receptor-positive neurons staining for PKCbetaII, decreased in denervated preparations. The same parameters related to PKCalpha, betaI or gamma remained unchanged. Overall, the present data strengthen the concept that mu-opioid receptors located on myenteric neurons are coupled to Ca(2+)-dependent PKCs. After chronic sympathetic denervation, a reduced efficiency of this coupling may predominantly involve PKCbetaII, although also PKCbetaI and gamma, but not PKCalpha, may be implicated.

The existence of a close relation between presynaptic inhibitory alpha(2)-adrenoceptor and mu-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on alpha(2)-adrenoceptors and mu-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of alpha(2)-adrenoceptors and mu-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of alpha(2)-adrenoceptor and mu-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of alpha(2)-adrenoceptors and mu-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions alpha(2)-adrenoceptor and mu-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Galpha(i1), Galpha(i2), Galpha(o) and Galpha(z) levels remained unchanged, while Galpha(i3) levels decreased, in the cortex; whereas, Galpha(i1), Galpha(i2) and Galpha(i3) levels significantly increased, and Galpha(o) and Galpha(z) levels remained unchanged, in the hippocampus. On the whole, the present data suggest that alpha(2)-adrenoceptor and mu-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed.

Supersensitivity to muscarinic, kappa- and mu-opioid agents modulating cholinergic neurons in the guinea pig colon develops after chronic sympathetic denervation. A possible role for protein kinase C (PKC) in contributing to development of these sensitivity changes was investigated. The PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced acetylcholine (ACh) overflow in preparations obtained from normal animals. The facilitatory effect of PMA was significantly reduced after prolonged exposure to the phorbol ester and by the PKC inhibitors, chelerythrine and calphostin C. Subsensitivity to the facilitatory effect of PMA developed after chronic sympathetic denervation. In this experimental condition, immunoblot analysis revealed reduced levels of PKC in myenteric plexus synaptosomes. The facilitatory effect of the muscarininc antagonist, scopolamine, on ACh overflow was significantly reduced by the phospolipase C (PLC) inhibitor, U73122, chelerythrine and calphostin C, both in normal and denervated animals. However, in both experimental groups, PLC antagonists and PKC antagonists did not affect the inhibitory effect of the muscarinic agonist, oxotremorine-M on ACh overflow. The inhibitory effects of U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on ACh overflow significantly increased in the presence of U73122, chelerythrine and calphostin C in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. These results indicate that activation of PKC enhances ACh release in the myenteric plexus of the guinea pig colon. At this level, chronic sympathetic denervation entails a reduced efficiency of the enzyme. In addition, PKC is involved in the inhibitory modulation of ACh release mediated by muscarinic-, kappa- and mu-opioid receptors, although with different modalities. Muscarinic receptors inhibit PKC activity, whereas kappa- and mu-opioid receptors increase PKC activity. Both the inhibitory and the facilitatory effect on PKC involve modulation of PLC activity. The possibility that the change in PKC activity represents one of the biochemical mechanisms at the basis of development of sensitivity changes to opioid and muscarinic agents after chronic sympathetic denervation is discussed.

The actions of adenosine, adenosine 5'-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), alpha,beta-methylene ATP (alpha,beta-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1) and P2X(2) receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y(1) mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP>/=2-MeSATP>alpha,beta-meATP>ATP=UTP=adenosine. This is suggestive of P2Y(1)-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to alpha,beta-meATP. As ATP and UTP are equipotent, the presence of a P2Y(2) receptor is indicated. ATP responses were inhibited by the P2Y(1)-selective antagonist MRS 2179, and suramin. P2Y(1) receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y(1) mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and N(omega)-nitro-L-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>alpha,betameATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X(2) receptors. Immunoreactivity to P2X(2) receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y(1) receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y(1), P2Y(2) and a novel P2Y receptor subtype responsive to alpha,beta-meATP in smooth muscle, while P2X(2) receptors mediate contraction of colonic smooth muscle.

We have studied the effects of naloxone on acetylcholine and noradrenaline release in the guinea-pig isolated distal colon, and have assessed the effect of naloxone on electrically-induced contractions of the longitudinal muscle and non-adrenergic, non-cholinergic (NANC) relaxations of the circular muscle coat. Naloxone dose-dependently increased resting and electrically-evoked acetylcholine release and electrically-evoked noradrenaline release. Naloxone was more potent in increasing resting acetylcholine release in colonic specimens obtained after chronic sympathetic denervation. Naloxone (1 μm) did not affect electrically-induced contractions of the longitudinal muscle, while it enhanced NANC relaxations of the circular muscle. The effects observed with naloxone in the present experiments suggest that opioid pathways exert a tonic restraint on neurotransmission in the guinea-pig colon. After suppression of the adrenergic inhibitory tone, the functional relevance of opioid pathways seems to be increased.

1 The present study examined the role of muscarinic receptors in the modulation of noradrenaline (NA) release in the guinea-pig isolated distal colon. The spontaneous endogenous NA overflow assayed by HPLC-ED was taken as an index of NA release from enteric noradrenergic nerve terminals. 2 Physostigmine (10 microM) significantly enhanced spontaneous endogenous NA overflow. Hyoscine (muscarinic antagonist), (R)-(-)-trihexyphenidyl and telenzepine (M1-selective antagonists), and 11[[2-[(diethylamino)methyl]-1-piperydil]acetyl]-5,11 -dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116, M2-selective antagonist) inhibited NA overflow in a concentration dependent manner, with the following EC50 values: 131.74 (18.19-953.96), 101.62 (58.83-175.60), 150 (60-330), 30 (5-170) nM, respectively. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M1- and M3-selective antagonist) had no significant effect up to 100 microM. 3 The muscarinic agonist oxotremorine inhibited NA overflow in a concentration dependent manner, with an EC50 value of 0.67 (0.30-1.51) microM. The response to oxotremorine was inhibited by muscarinic antagonists with the following order of potency: hyoscine = (R)-(-)-trihexyphenidyl = telenzepine > 4-DAMP >> AF-DX 116. 4 In the presence of 3 microM tetrodotoxin (TTX), the effect of oxotremorine and 4-DAMP was unchanged, while hyoscine, (R)-(-)-trihexyphenidyl, telenzepine and AF-DX 116, instead of inhibiting, significantly enhanced NA overflow. 5 The present results indicate that, in the guinea-pig colon, endogenous acetylcholine sustains spontaneous NA release by activating muscarinic receptors possibly located on interneurones. In addition, inhibitory muscarinic receptors may exist on adrenergic terminals.

Several calcium-channel blockers currently in use for the treatment of cardiovascular disorders have recently been tested for their effects on gastrointestinal motility. The rationale for this approach centers on the concept that calcium-channel blockers are at least as potent in inhibiting intestinal smooth muscle as in relaxing vascular smooth muscle. This review will give an outline of the most recent findings on the role of calcium and calcium channels in smooth muscle and neuronal function in the digestive system. It will also consider the mechanisms by which calcium-channel blockers may affect gastrointestinal motility and assess potential clinical applications in gastroenterology. The main goal for researchers in this field will be the development of gut-selective agents, with no cardiovascular side effects.

To assess the role of NMDA receptors in modulating neurotransmitter release in the myenteric plexus, we studied the effects of l -glutamic acid and NMDA on endogenous acetylcholine and noradrenaline overflow (assayed by HPLC) from the guinea pig isolated distal colon. l -Glutamic acid and NMDA enhanced electrically evoked acetylcholine and noradrenaline overflow and these effects were reversed by selective NMDA receptor antagonists. The possible functional significance of these findings was studied by measuring the efficiency of the colonic peristaltic reflex in the presence of NMDA receptor agonists. NMDA inhibited propulsion velocity at all concentrations tested, this effect being antagonized by (±)-2-amino-5-phosphonopentanoic acid and virtually abolished in sympathetically denervated animals. In conclusion, the inhibitory effect of NMDA on peristalsis, being almost entirely dependent on the integrity of sympathetic; pathways, could be, at least in part, due to NMDA-induced noradrenaline release.